Influence of filtering on the effective concentration and sterility of a 2% cyclosporine ophthalmic solution: a quality improvement perspective.

BACKGROUND: Pharmaceutical companies do not sell formulations for all diseases; thus, healthcare workers have to treat some diseases by concocting in-hospital preparations. An example is the high-concentration 2% cyclosporine A (CyA) ophthalmic solution. Utilizing a filter in sterility operations is a general practice for concocting in-hospital preparations, as is the case for preparing a 2% CyA ophthalmic solution. However, whether filtering is appropriate concerning the active ingredient content and bacterial contamination according to the post-preparing quality control of a 2% CyA ophthalmic solution is yet to be verified. METHODS: We conducted particle size, preparation concentration, and bacterial contamination studies to clarify aforementioned questions. First, we measured the particle size of CyA through a laser diffraction particle size distribution. Next, we measured the concentration after preparation with or without a 0.45-µm filter operation using an electrochemiluminescence immunoassay. Finally, bacterial contamination tests were conducted using an automated blood culture system to prepare a 2% CyA ophthalmic solution without a 0.45 µm filtering. Regarding the pore size of the filter in this study, it was set to 0.45 µm with reference to the book (the 6th edition) with recipes for the preparation of in-hospital preparations edited by the Japanese Society of Hospital Pharmacists. RESULTS: CyA had various particle sizes; approximately 30% of the total particles exceeded 0.45 µm. The mean ± standard deviation of filtered and non-filtered CyA concentrations in ophthalmic solutions were 346.51 ± 170.76 and 499.74 ± 76.95ng/mL, respectively (p = 0.011). Regarding bacterial contamination tests, aerobes and anaerobes microorganisms were not detected in 14 days of culture. CONCLUSIONS: Due to the results of this study, the concentration of CyA may be reduced by using a 0.45-µm filter during the preparation of CyA ophthalmic solutions, and furthermore that the use of a 0.45-µm filter may not contribute to sterility when preparing CyA ophthalmic solutions.

Metabolic control from the endolysosome: lysosome-resident amino acid transporters open novel therapeutic possibilities.

Amino acid transporters are generally recognized as machinery that transport amino acids from the extracellular environment into the cytoplasm. Although their primary function is the uptake of amino acids to supply the cell with nutrients and energy, endolysosome-resident amino acid (EL-aa) transporters possess several unique functions in accordance with their localization in intracellular vesicular membranes. They play pivotal roles in the maintenance of metabolic homeostasis via direct involvement in the amino acid sensing pathway, which regulates the activity of mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cellular metabolism. Additionally, some EL-aa transporters contribute to the maintenance of dynamic homeostasis of endolysosomes, including the regulation of endolysosomal acidity, by carrying amino acids out of endolysosomes. In addition, EL-aa transporters act as a scaffold to gather signaling molecules and multiple enzymes to control cellular metabolism on the endolysosomal membrane. Among EL-aa transporters, solute carrier family 15 member 4 (SLC15A4) is preferentially expressed in immune cells, including macrophages, dendritic cells, and B cells, and plays a key role in the integration of metabolic and inflammatory signals. In this review, we summarize our recent findings on EL-aa transporter contributions to inflammatory and metabolic signaling in the endolysosomes of immune cells by focusing on the SLC15 family, including SLC15A4 and SLC15A3, and discuss their uniqueness and universality. We also discuss the potential of targeting these EL-aa transporters in immune cells for the development of novel therapeutic strategies for inflammatory diseases. Because these transporters are highly expressed in immune cells and significantly alter the functions of immune cells, targeting them would provide a great advantage in ensuring a wide safety margin.

Determination of the Optimal Wavelength of the Hemolysis Index Measurement.

Many biochemical auto-analyzers have methods that measure the hemolysis index (HI) to quantitatively assess the degree of hemolysis. Past reports on HI are mostly in vitro studies. Therefore, we evaluated the optimal wavelength of HI measurement ex vivo using clinical samples. Four different wavelengths (410/451 nm: HI-1, 451/478 nm: HI-2, 545/596 nm: HI-3 and 571/596 nm: HI-4) were selected for HI measurement, and correlations were examined from the measurement results of 3890 clinical samples. Another set of 9446 clinical samples was used to examine the correlation of HI with lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and potassium (K). Strong correlations were found between HI-4 and HI-1 and between HI-4 and HI-3. HI-1 and HI-2 cannot correctly assess hemolysis for high bilirubin samples, and HI-3 cannot correctly assess hemolysis for high triglyceride samples. LDH, AST and K correlated positively with HI-4 in clinical samples. For every 1-unit increase in HI-4, LDH increased by 19.51 U/L, AST by 1.03 U/L and K by 0.061 mmol/L, comparable to reports of other studies. In clinical samples, HI-4 was less susceptible to bilirubin and chyle and reflected well the changes in LDH, AST and K caused by hemolysis. This suggested that the optimal wavelength for HI measurement is 571 nm.

Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique.

The purpose of this protocol is to screen and identify the physiologically relevant interactors of a lysosomal protein in living cells. Here, we describe how to identify solute carrier family 15 member 4 (SLC15A4)-interacting proteins by BioID and mass spectrometry analysis. This protocol utilizes fusion of SLC15A4 with a mutant form of biotin ligase, BirA. The fusion protein can promiscuously biotinylate the proteins proximal to SLC15A4. The biotinylated endogenous proteins are pulled down by magnetic streptavidin beads and detected by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2021).

Lysosomal amino acid transporters as key players in inflammatory diseases.

Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member A3 (SLC15A3) and member A4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

Exploration and time-serial validation of logistic regression models composed of multiple laboratory tests for early detection of HCV-associated hepatocellular carcinoma.

BACKGROUND: We developed a laboratory test-based regression model for early detection of hepatocellular carcinoma (HCC) associated with HCV in its surveillance. METHODS: This matched case-control study was conducted by enrolling 452 patients with chronic hepatitis and/or cirrhosis, including 129 patients complicated with HCC. One-to-one propensity score matching was performed by referring to sex, age, and fibrosis-4 index, which resulted in 102 patients each in HCC and non-HCC groups. Logistic regression models (LRM) for distinguishing the two groups were explored from variable combinations of laboratory tests. The model was validated by our new scheme of applying it retroactively to trimonthly previous datasets. RESULTS: Models with a practical level of diagnostic accuracy (C-statistic) were α-fetoprotein (AFP) alone (0.810), LRM3 comprising AFP, AST, and ALT (0.850), and LRM4 comprising AFP, AFP/(AST × ALT), and AST (0.862). After retroactive application of each model, LRM4 showed the highest distinction of the two groups at -12M, -6M, -3M with C-statistics of 0.654, 0.786, 0.834, respectively. LRM4 was accurate even after limiting cases to early-stage HCC. CONCLUSIONS: LRM4 was proved useful in prompting clinicians to perform timely image study in the surveillance. The retroactive validation scheme is applicable to assess diagnostic models of other neoplastic diseases.

Human SLC15A4 is crucial for TLR-mediated type I interferon production and mitochondrial integrity.

Solute carrier family 15 member 4 (SLC15A4) is an endolysosome-resident amino acid transporter that regulates innate immune responses, and is genetically associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and colitis. SLC15A4-deficient mice showed the amelioration of symptoms of these model diseases, and thus SLC15A4 is a promising therapeutic target of SLE and colitis. For developing a SLC15A4-based therapeutic strategy, understanding human SLC15A4's properties is essential. Here, we characterized human SLC15A4 and demonstrated that human SLC15A4 possessed pH- and temperature-dependent activity for the transportation of dipeptides or tripeptides. Human SLC15A4 localized in LAMP1+ compartments and constitutively associated with Raptor and LAMTORs. We also investigated SLC15A4's role in inflammatory responses using the human plasmacytoid dendritic cell line, CAL-1. Knock down (KD) of the SLC15A4 gene in CAL-1 (SLC15A4-KD CAL-1) impaired Toll-like receptor (TLR) 7/8 or TLR9-triggered type I interferon (IFN-I) production and mTORC1 activity, indicating that human SLC15A4 is critical for TLR7/8/9-mediated inflammatory signaling. We also examined SLC15A4's role in the autophagy response since SLC15A4 loss caused the decrease of mTORC1 activity, which greatly influences autophagy. We found that SLC15A4 was not required for autophagy induction, but was critical for autophagy sustainability. Notably, SLC15A4-KD CAL-1 severely decreased mitochondrial membrane potential in starvation conditions. Our findings revealed that SLC15A4 plays a key role in mitochondrial integrity in human cells, which might benefit immune cells in fulfilling their functions in an inflammatory milieu.

What is the best wavelength for the measurement of hemolysis index?

BACKGROUND: Hemolysis is a common problem in the handling of serum specimens. The hemolysis index (HI) provides a warning of hemolysis in auto-analyzers. However, HI has not been standardized, and each laboratory's original method is applied. Especially, the wavelength used for HI measurement is different in each laboratory. Thus, we investigated the warning ability of HI at various wavelengths. METHODS: We selected 4 wavelength types, and each HI was measured and calculated (410 nm/HI-1, 451 nm/HI-2, 545 nm/HI-3, and 571 nm/HI-4). To compare the 4 HI types, we investigated the influence of 3 interference components using artificially hemolyzed specimens (AHSs). We also investigated both the relationship between HI and hemoglobin concentration (Hb) and that between HI and 31 biochemical test values in AHSs. RESULTS: In the interference assessment, only HI-4 showed no influence on the 3 interference components. The correlation between Hb and HI-4 was very strong (rS = 0.9987). A 1-unit increase in HI-4 corresponded to a 14.8-mg/dL increase in Hb. CONCLUSION: We found the best wavelength for HI to be at or near 571 nm.

Type I interferon limits mast cell-mediated anaphylaxis by controlling secretory granule homeostasis.

Type I interferon (IFN-I) is a family of multifunctional cytokines that modulate the innate and adaptive immunity and are used to treat mastocytosis. Although IFN-I is known to suppress mast cell function, including histamine release, the mechanisms behind its effects on mast cells have been poorly understood. We here investigated IFN-I's action on mast cells using interferon-α/ß receptor subunit 1 (Ifnar1)-deficient mice, which lack a functional IFN-I receptor complex, and revealed that IFN-I in the steady state is critical for mast cell homeostasis, the disruption of which is centrally involved in systemic anaphylaxis. Ifnar1-deficient mice showed exacerbated systemic anaphylaxis after sensitization, which was associated with increased histamine in the circulation, even though the mast cell numbers and high affinity immunoglobulin E receptor (FcεRI) expression levels were similar between Ifnar1-deficient and wild-type (WT) mice. Ifnar1-deficient mast cells showed increased secretory granule synthesis and exocytosis, which probably involved the increased transcription of Tfeb. Signal transducer and activator of transcription 1(Stat1) and Stat2 were unexpectedly insufficient to mediate these IFN-I functions, and instead, Stat3 played a critical role in a redundant manner with Stat1. Our findings revealed a novel regulation mechanism of mast cell homeostasis, in which IFN-I controls lysosome-related organelle biogenesis.

Lysosome biogenesis regulated by the amino-acid transporter SLC15A4 is critical for functional integrity of mast cells.

Mast cells possess specialized lysosomes, so-called secretory granules, which play a key role not only in allergic responses but also in various immune disorders. The molecular mechanisms that control secretory-granule formation are not fully understood. Solute carrier family member 15A4 (SLC15A4) is a lysosome-resident amino-acid/oligopeptide transporter that is preferentially expressed in hematopoietic lineage cells. Here, we demonstrated that SLC15A4 is required for mast-cell secretory-granule homeostasis, and limits mast-cell functions and inflammatory responses by controlling the mTORC1-TFEB signaling axis. In mouse Slc15a4-/- mast cells, diminished mTORC1 activity increased the expression and nuclear translocation of TFEB, a transcription factor, which caused secretory granules to degranulate more potently. This alteration of TFEB function in mast cells strongly affected the FcεRI-mediated responses and IL-33-triggered inflammatory responses both in vitro and in vivo. Our results reveal a close relationship between SLC15A4 and secretory-granule biogenesis that is critical for the functional integrity of mast cells.

Elevated Soluble Platelet Glycoprotein VI Levels in Patients After Living Donor Liver Transplantation.

Plasma-soluble platelet glycoprotein VI (sGPVI) levels were examined in patients undergoing living donor liver transplantation (LDLT), and the relationship between platelet activation and thrombocytopenia was evaluated to understand the mechanism of thrombocytopenia in LDLT. Platelet counts were significantly higher in the donors compared to the recipient, and the plasma sGPVI levels increased in both groups after the operation. Regarding the relationship between the platelet counts and the sGPVI levels, the slope varied on different days, and it became negative on day 3, suggesting that the plasma sGPVI levels are related to platelet activation in LDLT. The frequency of complications was high in the nonsurvivors. The platelet counts were higher in the survivors than in the nonsurvivors on days 14 and 28. Although the plasma levels of sGPVI in the survivors increased after the operation, those in the nonsurvivors were high only on day 3. Although the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 domain, member 13) levels were markedly reduced, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were markedly elevated during LDLT. The antithrombin activity was significantly lower (day 14) and VWFpp (day 28) was significantly higher in the nonsurvivors than in the survivors. These findings suggest that platelet activation first occurs after LDLT, and it is high in the nonsurvivors on day 3. Thereafter, the hemostatic abnormality and vascular endothelial cell injuries may appear on days 14 and 28.

C4b binding protein negatively regulates TLR1/2 response.

TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.

Inflammatory responses increase secretion of MD-1 protein.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6.

Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.

The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production.

SLC15A4 is a lysosome-resident, proton-coupled amino-acid transporter that moves histidine and oligopeptides from inside the lysosome to the cytosol of eukaryotic cells. SLC15A4 is required for Toll-like receptor 7 (TLR7)- and TLR9-mediated type I interferon (IFN-I) productions in plasmacytoid dendritic cells (pDCs) and is involved in the pathogenesis of certain diseases including lupus-like autoimmunity. How SLC15A4 contributes to diseases is largely unknown. Here we have shown that B cell SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Our findings revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production.

Prolonged thrombocytopenia after living donor liver transplantation is a strong prognostic predictor irrespective of splenectomy: the significance of ADAMTS13 and graft function [corrected].

The precise mechanism of prolonged thrombocytopenia following living donor liver transplantation (LDLT) remains unclear. To determine risk factors associated with prolonged thrombocytopenia following LDLT, with a focus on the activity of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs member 13) and the influence of splenectomy. Adult LDLT patients were divided into two groups on the basis of platelet counts (100 × 10(3)/µL) on POD 14: high and low platelet (HP and LP) groups. Survival analysis was performed in the 100 patients, and ADAMTS13 activity and von Willebrand factor (VWF) levels in the plasma were measured in 65 adult recipients. The 6-month survival rate was significantly lower in the LP group (n = 36) than in the HP group (n = 62) (61.1 vs. 93.5 %). ADAMTS13 activity had been significantly lower in the LP group (n = 23) than in the HP group (n = 42). The VWF/ADAMTS13 ratio was significantly higher in the LP group than in the HP group. The independent risk factors for thrombocytopenia on POD14 were preoperative AT levels and ADAMTS13 activity on POD14. TPO levels on POD14 were significantly higher in the LP group than in the HP group, while those on POD28 in the LP group were significantly decreased, despite the low platelet levels. Irrespective of splenectomy, platelet counts and ADAMTS13 activity in the LP group remained low until POD28, while VWF/ADAMTS13 ratio significantly increased until POD28. These results suggest that prolonged thrombocytopenia after LDLT was associated with not only a decrease in ADAMTS13 due to sinusoidal endothelial cell injury, but also low TPO production due to hepatocyte dysfunction, irrespective of splenectomy [corrected].

How do cells optimize luminal environments of endosomes/lysosomes for efficient inflammatory responses?

The endosome/lysosome compartments play pivotal roles in immune cell functions as signalling platforms. These intracellular compartments can efficiently restrict the localization of signalling complexes and temporally regulate signalling events to produce qualitatively different outcomes. Immune cells also exploit the endosome/lysosome system for signal transduction and intercellular communication to elicit immune responses. Antigen-presenting cells such as dendritic cells and macrophages take up pathogens by endocytosis and prepare antigens via the endosome/lysosome system. At the same time, pathogen-derived DNA and RNA are recognized by immune sensors at the endosome/lysosome compartments, which transmit signals to induce immune responses. Recent studies revealed the importance of controlling the endosomal/lysosomal environment for eliciting efficient signalling events at the endosomes/lysosomes. Many factors including pH, membrane potential, amino acid concentrations and lipid composition are finely tuned at the endosome/lysosome compartments, and dysregulation of these factors greatly affect immune cell functions. Redox-related molecules and various types of transporters are involved in the control of endosomal/lysosomal environment and could be good therapeutic targets for treating autoimmune diseases.

Specific mtDNA mutations in mouse carcinoma cells suppress their tumor formation via activation of the host innate immune system.

In mammalian species, mitochondrial DNA (mtDNA) with pathogenic mutations that induce mitochondrial respiration defects has been proposed to be involved in tumor phenotypes via induction of enhanced glycolysis under normoxic conditions (the Warburg effects). However, because both nuclear DNA and mtDNA control mitochondrial respiratory function, it is difficult to exclude the possible contribution of nuclear DNA mutations to mitochondrial respiration defects and the resultant expression of tumor phenotypes. Therefore, it is important to generate transmitochondrial cybrids sharing the same nuclear DNA background but carrying mtDNA with and without the mutations by using intercellular mtDNA transfer technology. Our previous studies isolated transmitochondrial cybrids and showed that specific mtDNA mutations enhanced tumor progression as a consequence of overproduction of reactive oxygen species (ROS). This study assessed whether mtDNA mutations inducing ROS overproduction always enhance tumor progression. We introduced mtDNA from senescence-accelerated mice P1 (SAMP1) into C57BL/6J (B6) mice-derived Lewis lung carcinoma P29 cells, and isolated new transmitochondrial cybrids (P29mtSAMP1 cybrids) that overproduced ROS. The inoculation of the cybrids into B6 mice unexpectedly showed that mtDNA from SAMP1 mice conversely induced tumor suppression. Moreover, the tumor suppression of P29mtSAMP1 cybrids in B6 mice occurred as a consequence of innate immune responses of the host B6 mice. Enzyme pretreatment experiments of P29mtSAMP1 cybrids revealed that some peptides encoded by mtDNA and expressed on the cell surface of P29mtSAMP1 cybrids induce increased IL-6 production from innate immune cells (dendritic cells) of B6 mice, and mediate augmented inflammatory responses around the tumor-inoculated environment. These observations indicate presence of a novel role of mtDNA in tumor phenotype, and provide new insights into the fields of mitochondrial tumor biology and tumor immunology.