Pharmaceutical composition for improving inflammation caused by chronic hypoxic condition

Abstract

To provide a pharmaceutical composition that improves exacerbation of an inflammatory reaction due to a chronic hypoxic condition resulting from reduced circulatory or respiratory function, or from anemia.SOLUTION: It has been found that pyridoxal synthase Pyridoxamine-5'-phosphate oxidase (PNPO) functions as a novel oxygen-sensing mechanism. A pharmaceutical composition containing pyridoxal or pyridoxal phosphate as an active ingredient acts as a pharmaceutical composition for improving exacerbation of an inflammatory reaction due to a chronic hypoxic condition.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present inventors have found that chronic hypoxia exacerbates inflammatory responses. Furthermore, they have found that the biological response under chronic hypoxia is independent of the hypoxia response factor (HIF) pathway. Based on this finding, the present invention relates to a pharmaceutical composition for ameliorating chronic hypoxia and the resulting inflammatory responses. [Background technology]

[0002] For metazoans on Earth, oxygen is a gas molecule essential for survival and affects various physiological processes. The HIF pathway is known as a system that detects acute hypoxia. HIF (Hypoxia Inducible Factor) is a protein that is induced when cells fall into a hypoxic state and functions as a transcription factor, enhancing the transcription of various genes and helping cells escape from hypoxic conditions (Non-Patent Document 1). However, the cellular response to chronic hypoxia has not yet been elucidated.

[0003] To analyze the cellular response to chronic hypoxia, we used mice with renal anemia under chronic hypoxia and found that chronic hypoxia exacerbates inflammation and that the cellular response to chronic hypoxia is independent of the HIF pathway.

[0004] Chronic hypoxia occurs in peripheral tissues due to cardiovascular diseases such as heart failure associated with aging and respiratory dysfunction such as chronic respiratory failure. Additionally, underlying diseases such as old age, smoking, obesity, and cardiovascular disease, which are cited as major risk factors for the cytokine storm caused by SARS-CoV-2 infection and the resulting severe illness, are also associated with chronic hypoxia. Suppressing the exacerbation of the inflammatory response resulting from chronic hypoxia can significantly reduce the risk of severe illness. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaelin, WG, Ratcliffe, Jr, PJ, 2008, Mol. Cell, Vol.30, pp.393-402,doi:10.1016 / j.molcel.2008.04.009 [Non-patent document 2] Yamazaki, S., et al., Nat. Commun., 2013, 4, 1950,doi:10.1038 / ncomms2950 [Non-patent document 3] Souma, T., et al., J. Am. oc. Nephrol., 2016, Vol.27, pp.428-438,doi:10.1681 / ASN.2014121184 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a pharmaceutical composition that ameliorates inflammation caused by chronic hypoxia, based on findings obtained from the analysis of cellular responses under chronic hypoxia. [Means for solving the problem]

[0007] The present invention relates to the following pharmaceutical compositions. (1) A pharmaceutical composition for suppressing inflammation caused by chronic hypoxia, the pharmaceutical composition comprising, as an active ingredient, a compound that enhances lysosomal function. The present inventors have demonstrated that chronic hypoxia leads to inflammatory polarization of macrophages due to decreased lysosomal activity, leading to exacerbation of inflammation. Therefore, enhancing lysosomal function can suppress inflammation caused by chronic hypoxia.

[0008] (2) The pharmaceutical composition according to (1), wherein the active ingredient is activated vitamin B6. When we investigated the decline in lysosomal activity, we found that the active form of vitamin B6 was significantly reduced. By supplementing the active form of vitamin B6, lysosomal activity can be restored and inflammation can be suppressed.

[0009] (3) The pharmaceutical composition according to (1) or (2), wherein the chronic hypoxic state is caused by a decrease in circulatory or respiratory function or anemia. Chronic hypoxia exacerbates inflammation, and if an inflammatory disease occurs concomitantly, the condition often becomes severe. When a patient is thought to be in a chronic hypoxic state and develops an inflammatory disease, administering activated vitamin B6, which restores lysosomal function, is effective. [Brief explanation of the drawings]

[0010] [Figure 1] (A) A graph showing that chronic hypoxia model mice are highly susceptible to DSS (dextran sulfate sodium)-induced colitis. Genetically modified inherited super-anemic mice (ISAM) and control mice were given drinking water containing 3% DSS, and weight changes over time were recorded. (B) and (C) Graphs showing colon shortening 6 days after colitis was induced with 3% DSS. (B) shows the appearance of the colon, and (C) shows its length. (D) HE-stained image of a tissue section. (E) Histological score. [Figure 2] (A) Gene expression in the colon 6 days after DSS administration. (B) Gene expression analysis of macrophages obtained from peritoneal exudate treated with LPS (lipopolysaccharide). (C) Cytokines in the culture supernatant of macrophages obtained from peritoneal exudate were analyzed by ELISA. [Figure 3](A) Schematic diagram of the experimental design in which bone marrow-derived macrophages (BMDMs) were cultured under different oxygen tensions and an inflammatory response was induced with LPS. (B) Heat map showing gene changes after LPS stimulation of BMDMs cultured under the conditions shown in (A). (C) Diagram showing the results of cytokine analysis in the BMDM culture supernatant 12 hours after stimulation with LPS. (D) Diagram showing the results of enrichment analysis (Enrichr) of genes with reduced expression. [Figure 4] (A) Microscopic images showing the analysis of lysosomal acidification by AcidiFluor ORANGE staining in BMDMs cultured under normal or hypoxic conditions after stimulation with LPS. Representative images are shown for each condition. (B) Immunoblotting analysis of Lamp1 protein expression. (C) Microscopic images (left) and quantitative results (right) showing the analysis of lysosomal activity under long-term hypoxia after Hif1α deletion using AcidiFluor ORANGE staining. (D) Heat map showing the expression of lysosomal-related genes associated with lysosomal acidification. Gene expression in BMDMs cultured under normoxia in the presence of lysosomal inhibitors is shown. (E) Scatter plot showing the correlation between the fold change in gene expression (AUC ratio) induced by LPS stimulation in BMDMs under long-term hypoxia and the administration of a lysosomal inhibitor (ConA). The strength of the correlation was evaluated using Pearson's product-moment correlation coefficient. [Figure 5](A) Volcano plot comparing the metabolome of bone marrow-derived macrophages cultured under chronic hypoxic conditions (chronic hypoxia-BMDMs: CHyp-BMDMs) and normoxia-BMDMs (normoxia-BMDMs: Norm-BMDMs). The horizontal dashed line indicates the significance threshold (p = 0.01), and the vertical dashed line indicates a 2-fold or greater increase or decrease. P values ​​were calculated using Welch's t-test. (B) Relative levels of vitamin B6 derivatives in BMDMs. Metabolites were measured on day 3 of differentiation and after LPS stimulation. (C) Relative concentrations of pyridoxal phosphate in serum of ISAMs. (D) Relative levels of pyridoxal phosphate in hypoxic mouse lung tissue. (E) Relative levels of pyridoxal phosphate in BMDMs. (F) Lysosomal acidification in BMDMs. Representative AcidiFluor ORANGE staining images (left) and quantification results (right) under each condition are shown. [Figure 6] (A) Relative pyridoxal phosphate levels in BMDMs. (B) Schematic diagram of vitamin B6 metabolism and catalytic enzymes. (C) Immunoblot analysis of PNPO (pyridoxine / pyridoxamine 5'-phosphate oxidase) protein expression. (D) Time course of pyridoxal phosphate levels after PNPO knockdown with siRNA, culture under 1% O2, and reoxygenation. [Figure 7] (A) Microscopic image of intracellular ferrous iron in BMDMs stained with FerroOrange. (B) Analysis of gene expression induced by LPS stimulation in BMDMs differentiated in the presence of a 2-oxoglutarate (2OG)-dependent dioxygenase inhibitor. (C) Immunoblot analysis of 2OG-dependent dioxygenase protein expression in BMDMs. (D) ChIP assay detecting binding of acetylated histone H3K27 in BMDMs. [Figure 8](A) Microscopic image of intracellular ferrous iron in BMDMs stained with FerroOrange. Hoechst33342 was used for nuclear staining. A representative staining image (top) and a graph quantifying the fluorescence intensity of FerroOrange (bottom) are shown. (B) Graph showing time-dependent gene expression in BMDMs after LPS stimulation. (C) Graph showing 2OG-dependent dioxygenase protein expression by immunoblot analysis. [Figure 9] (A) Microscopic images of lysosomal acidification in BMDMs stained with AcidiFluor ORANGE. Representative staining images for each condition are shown. (B) Immunoblotting analysis of Lamp1 protein expression. (C) Immunoblotting analysis of lysosome-related gene expression. (D) Immunoblotting analysis of TET2 protein expression. (E) Il6 expression in BMDMs after LPS stimulation. (F) ELISA analysis of IL-6 in the culture supernatant of BMDMs. (G) Il6 expression in lung tissue from mice exposed to hypoxia for 3 days. (H) Relative amounts of pyridoxal phosphate (left) in lung tissue from mice administered pyridoxal during hypoxia exposure and Il6 expression levels 4 hours after LPS administration. [Figure 10] A schematic diagram showing the mechanism for sensing chronic hypoxia and the mechanism for controlling the inflammatory response of macrophages. DETAILED DESCRIPTION OF THE INVENTION

[0011] As described above, the present inventors have discovered that chronic hypoxia exacerbates inflammation and have further clarified the mechanism by which hypoxia is sensed, thereby completing the present invention. Chronic hypoxia can be caused by a variety of factors, including chronic respiratory diseases such as chronic obstructive pulmonary disease and interstitial pneumonia, cardiovascular diseases such as heart failure, and anemia. As will be explained in detail below, it has been revealed that the exacerbation of inflammation caused by chronic hypoxia can be improved by active vitamin B6. Therefore, a pharmaceutical composition containing active vitamin B6 as an active ingredient can be used as a therapeutic agent for inflammatory diseases caused by chronic hypoxia.

[0012] In addition, activated vitamin B6 can suppress the exacerbation of inflammatory diseases and prevent them from becoming severe, even when patients in a chronic hypoxic state develop inflammatory diseases due to infectious diseases, etc. For patients who are thought to be in a chronic hypoxic state due to respiratory diseases, circulatory diseases, etc., taking activated vitamin B6 as a medicine or supplement is also useful for preventing the exacerbation of inflammatory diseases.

[0013] Active vitamin B6 includes pyridoxal and pyridoxal phosphate (pyridoxal-5'-phosphate), as well as pharmaceutically acceptable salts, ester derivatives, and solvates. Active vitamin B6 preparations have already been administered to patients with vitamin B6 deficiency, and their safety and administration methods have been established. For example, pyridoxal phosphate, as pyridoxal phosphate ester hydrate, is already used as an injection for subcutaneous, intravenous, and intramuscular injection, as well as an oral preparation. Active vitamin B6 is typically administered to adults at a dose of 5-60 mg, one to three times daily, by injection or orally. Alternatively, low doses may be taken prophylactically as a supplement. The dosage can, of course, be adjusted according to age and symptoms.

[0014] The pharmaceutical composition of the present invention may be mixed with pharmaceutically inert inorganic or organic excipients. Suitable excipients for tablets, sugar-coated tablets, or hard gelatin capsules include lactose, corn starch or its derivatives, talc, or stearic acid or its salts. Suitable excipients for soft gelatin capsules include vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. Examples of excipients for preparing solutions and syrups include water, polyols, sucrose, invert sugar, and glucose. Examples of excipients for injections include water, alcohol, polyols, glycerin, and vegetable oils. The composition may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for changing osmotic pressure, buffers, coating agents, or antioxidants. Furthermore, it can be administered in combination with other therapeutically useful drugs, for example, medicines for diseases that cause chronic hypoxia, i.e., drugs for treating chronic respiratory diseases or cardiovascular diseases, or anti-inflammatory drugs, or can be mixed with ingredients effective for these diseases to form a pharmaceutical composition. Note that in the following, the term "chronic hypoxic state" is used mainly for human diseases and mouse models, while "long-term hypoxic state" is used in cell experiments, but the terms can be used interchangeably.

[0015] [Relationship between chronic hypoxia and exacerbated inflammation] First, we demonstrate that chronic hypoxia exacerbates inflammation through experiments using a disease model. Genetically engineered mice with renal anemia (ISAM) (Non-Patent Documents 2 and 3) were used as a model of chronic hypoxia. Age-matched ISAM and control mice (5–6 months of age) were given 3% dextran sulfate sodium (DSS, MP Biomedicals) in their drinking water ad libitum for 6 days to induce colitis. ISAM mice showed significant weight loss compared to controls, and on day 5 of DSS intake, significant weight loss was observed compared to controls (Figure 1A). On day 6 of colitis induction, mice were dissected for analysis, and colon length was measured. The ISAM mice had significantly shorter colons than control mice (Figures 1B and 1C). Colons were fixed overnight at 4°C in Mildform 10N (Fujifilm Wako Pure Chemical Industries), sectioned, and subjected to pathological analysis, revealing significant histopathological damage (Figures 1D and 1E). In other words, ISAMs in a hypoxic state due to renal anemia are thought to be more sensitive to DSS and to have more severe inflammation than control mice.

[0016] We analyzed the expression of inflammatory cytokines in the colonic tissue of mice with colitis induction by RT-PCR. RNA was extracted from the colonic tissue of mice on day 6 of DSS ingestion using standard methods, cDNA was synthesized, and the expression of inflammatory cytokines was analyzed by real-time PCR. In ISAM mice with colitis induced with DSS (Fig. 2A, ID), the expression of Il6, Il23, and Il1b was significantly higher than that in colitis-induced control mice (Fig. 2A, CD), whereas the expression of Il12b and TNFα was similar (Fig. 2A).

[0017] We also analyzed the expression of inflammatory cytokines expressed in macrophages. First, to collect macrophages from peritoneal exudates, 2 mL of 4% thioglycollate was injected intraperitoneally. After 3 days, cells were isolated from the exudates. The isolated cells were incubated in culture plates for 2 hours, then washed with PBS. Adherent cells were used for experiments. They were stimulated with 100 ng / mL LPS, and the expression of Il6, Il1b, and Il10 was analyzed after 2 and 4 hours. Macrophages obtained from the peritoneal exudates of ISAM mice showed significantly higher expression of the pro-inflammatory cytokines Il6 and Il1b, but lower expression of the anti-inflammatory cytokine Il10 (Figure 2B).

[0018] Macrophage culture supernatants were also analyzed by ELISA. Macrophages were stimulated with 100 ng / mL LPS, and IL-6 levels were measured after 12 or 24 hours using an ELISA kit (R&D Systems). IL-1β levels were measured after 2 hours of incubation with 1 mM ATP (Figure 2C). After LPS stimulation, macrophage culture supernatants obtained from ISAMs secreted significantly higher levels of IL-6 and IL-1β than controls.

[0019] These results suggest that ISAM macrophages generate a stronger inflammatory response than control macrophages. Although not shown here, cytokine gene expression in bone marrow-derived macrophages differentiated under normoxia was comparable between ISAM and control mice. Therefore, the inflammatory phenotype of ISAM macrophages is thought to be enhanced under hypoxia.

[0020] [Study using bone marrow-derived macrophages] To examine the effect of oxygen tension on the inflammatory phenotype of macrophages, bone marrow-derived macrophages (BMDMs) were prepared by differentiation under normoxia (20%) and 1% oxygen. Macrophages differentiated under normoxia were designated Norm-BMDMs, while those differentiated under 1% oxygen were designated CHyp-BMDMs. Norm-BMDMs and CHyp-BMDMs were stimulated with LPS under each oxygen concentration. Furthermore, cells differentiated under normoxia and cultured under 1% oxygen for 12 hours were stimulated with LPS and designated AHyp-BMDMs as an acute hypoxic model (Figure 3A).

[0021] We performed transcriptome analysis of the LPS-induced inflammatory response in the three types of BMDMs by RNA-seq (Figure 3B). LPS stimulation increased the expression of typical HIF target genes in AHyp-BMDMs and CHyp-BMDMs compared with Norm-BMDMs. Furthermore, CHyp-BMDMs exhibited a significant increase in the expression of pro-inflammatory genes and a decrease in the expression of anti-inflammatory genes, whereas AHyp-BMDMs did not. Indeed, ELISA analysis revealed that LPS stimulation resulted in significantly higher secretion of IL-6 and TNF-α in CHyp-BMDMs than in Norm-BMDMs (Figure 3C).

[0022] Next, we investigated whether the HIF pathway was involved in gene expression changes in CHyp-BMDMs. In our experimental system, HIF-2α was barely detected regardless of oxygen concentration, so we investigated the contribution of HIF1α. F / F Mouse (B6.129-Hif1a tm3RsjoB6.Cg-Tg(Tek-cre)12Flv / J) and TIE2-Cre mice (B6.Cg-Tg(Tek-cre)12Flv / J) were purchased from the Jackson Laboratory and crossed with HIF1α-deficient macrophages. Although data are not shown here, HIF1α deficiency did not alter inflammatory gene expression in CHyp-BMDMs, except for Il1b, which is regulated by HIF-1α. Therefore, we concluded that the regulation of inflammatory genes in BMDMs under long-term hypoxia is not mediated by the PHD-HIF pathway.

[0023] The total amount of transcripts was analyzed over time up to 24 hours after LPS stimulation. BMDMs were cultured under long-term or acute hypoxia and then stimulated with LPS. Genes were classified into seven groups: those with increased or decreased expression, those with common increased or decreased expression, and those with no change in expression. Pathway analysis of the highly and poorly expressed genes was then performed (Figure 3D). There were gene groups with chronic hypoxia-specific, acute hypoxia-specific, or common decreased expression, suggesting that long-term hypoxia suppresses lysosomal activity.

[0024] [Decreased lysosomal activity due to chronic hypoxia] To analyze lysosomal activity under chronic hypoxia, we analyzed lysosomal acidification. Lysosomal acidification was examined by staining with the AcidiFluor ORANGE kit (Goryo Chemical) and observing lysosomal acidification under a confocal microscope. Fluorescence intensity was measured using LASX software (Leica). As expected, fluorescence intensity, indicating lysosomal acidification, was reduced in CHyp-BMDMs (Figure 4A). Furthermore, we analyzed the expression of the lysosomal membrane protein Lamp1 by immunoblotting. The results showed that the expression of Lamp1, a lysosomal membrane protein, was reduced in CHyp-BMDMs (Figure 4B). Lysosomal acidification was also measured in BMDMs derived from Hif1α-deficient mice (Hif1αF / F:Tie2-Cre) as described in Figure 4A. Similarly, reduced lysosomal acidification was observed in BMDMs derived from Hif1α-deficient mice (Figure 4C). Lysosomal acidification under prolonged hypoxia was also suppressed in Hif1α-deficient mice, indicating that HIF activity is not required for the lysosomal response to chronic hypoxia.

[0025] To compare lysosomal inhibition and long-term hypoxia, we treated Norm-BMDMs with lysosomal inhibitors (concanamycin A, bafilomycin A1) and analyzed LPS-induced transcriptome changes by RNA-seq (Figure 4D). Lysosomal-related genes, which were also reduced by concanamycin A (ConA) and bafilomycin A1 treatment, were also reduced in CHyp-BMDMs, as confirmed separately, consistent with the effects of the two lysosomal inhibitors. A scatter plot of the correlation between long-term hypoxia and the gene expression changes (AUC ratio) induced by the lysosomal inhibitor (ConA) in BMDMs revealed that the effect of the lysosomal inhibitor was positively correlated with long-term hypoxia (Figure 4E). GSEA analysis revealed that long-term hypoxia and lysosomal inhibitors had strikingly similar effects on LPS-induced transcripts in macrophages. These results suggest that lysosomal inhibition due to chronic hypoxia contributes to the proinflammatory phenotype of macrophages.

[0026] [Lysosomal regulation mechanisms] To gain insight into lysosomal regulation, we performed metabolomic analysis of Norm-BMDMs, CHyp-BMDMs, and BMDMs differentiated under 5% oxygen. Comparing the metabolites of CHyp-BMDMs and Norm-BMDMs revealed statistically significant differences in five metabolites (Figure 5A). Among these, pyridoxal (PL) and pyridoxal phosphate (PLP) were significantly decreased in CHyp-BMDMs. Furthermore, we measured the levels of pyridoxal and pyridoxal phosphate after LPS stimulation under 5% and 1% oxygen tensions. The decrease in pyridoxal and pyridoxal phosphate was dependent on the oxygen tension. In contrast, pyridoxine, a major source of pyridoxal and pyridoxal phosphate in the culture medium, did not change with oxygen conditions (Figure 5B). The decrease in pyridoxal phosphate was also observed in the serum of ISAMs (Figure 5C) and in the lung tissue of mice exposed to hypoxia (7% O2) for 3 days (Figure 5D). Furthermore, when cultured in pyridoxine-free medium, lysosomal acidification was suppressed in Norm-BMDMs to the same extent as in CHyp-BMDMs (Figures 5E and 5F). These results suggest that the reduction of pyridoxal phosphate during prolonged hypoxia suppresses lysosomal activity.

[0027] Recently, it has been reported that HIF1 upregulates the expression of pyridoxal phosphatase (PDXP), resulting in the degradation of pyridoxal phosphate. Therefore, we analyzed whether pyridoxal phosphate levels are regulated by HIF1 in BMDMs derived from HIF1-deficient mice (Figure 6A). Under long-term hypoxia (CHyp-BMDMs), pyridoxal phosphate levels decreased regardless of the status of Hif1α, indicating that the decrease in pyridoxal phosphate induced by chronic hypoxia is independent of HIF1α.

[0028] Because pyridoxine / pyridoxamine 5'-phosphate oxidase (PNPO) requires O2 as a substrate, prolonged hypoxia is expected to inhibit the catalytic reaction of PNPO, resulting in the depletion of pyridoxal phosphate and pyridoxal (Figure 6B). Using U937 cells in which PNPO was knocked down with siRNA (Figure 6C), we analyzed the contribution of PNPO to oxygen-dependent pyridoxal phosphate synthesis. After exposure to 1% O2 and subsequent reoxygenation, control cells showed a rapid increase in pyridoxal phosphate, whereas the increase in pyridoxal phosphate was significantly slower in PNPO-knocked down cells (Figure 6D). These results suggest that PNPO functions as an oxygen sensor regulating pyridoxal phosphate synthesis. The siRNA and control RNA used for PNPO knockdown were purchased from Sigma and transfected using GenomONE-SI (Ishihara Sangyo Kaisha).

[0029] Staining of intracellular ferrous iron using FerroOrange (Dojindo Laboratories) revealed a significant decrease in intracellular ferrous iron in CHyp-BMDMs (Figure 7A). Chronic hypoxia is predicted to result in a decrease in ferrous iron-dependent enzyme activity, leading to an inflammatory phenotype. Therefore, we analyzed 2-oxoglutarate (2OG)-dependent dioxygenase as a possible ferrous iron-dependent protein responsible for the inflammatory phenotype (Figure 7B). BMDMs were cultured under normoxia in the presence of a 2OG-dependent dioxygenase inhibitor, stimulated with LPS, and the expression of inflammatory cytokines was analyzed by RT-PCR. The 2OG-dependent dioxygenase inhibitors used were as follows: PHD inhibitor: GSK360A (AdipoGen life science), KDM6A inhibitor: GSKJ4 (TOCRIS Bioscience), KDM5 inhibitor: KDM5-C70 (Xcess Biosciences), TET inhibitor: Bobcat339 (InvivoChem).

[0030] TET inhibitors induced a pattern of Il6, Tnfa, and Il10 gene expression similar to that observed under chronic hypoxia. Furthermore, activation of Il1b in CHyp-BMDMs was similar to that observed under PHD inhibition. Because TET2 is primarily expressed in BMDMs, we focused on TET2 to analyze its function. Immunoblot analysis revealed that TET2 protein significantly accumulated in Norm-BMDMs in response to LPS, whereas it was barely detectable in CHyp-BMDMs despite LPS-induced increases in TET2 mRNA (Figure 7C). Because TET2 has a high Km for ferrous iron, it is likely to lose ferrous iron and become unstable under chronic hypoxia. To detect acetylated histones, we performed ChIP assays targeting enhancer regions of inflammatory cytokines using an anti-histone H3K27ac antibody (MAB Institute). After LPS treatment, CHyp-BMDMs showed increased acetylated histone H3K27 at the Il6 enhancer compared to Norm-BMDMs (Fig. 7D). These results suggest that CHyp-BMDMs share a phenotype similar to that of previously reported Tet2-deficient macrophages.

[0031] We analyzed the effect of lysosomal activity inhibition on intracellular iron (II). Lysosomes were inhibited by ConA treatment, and intracellular iron (II) was stained with Ferro Orange (Fig. 8A). Inhibition of lysosomal activity by ConA treatment reduced intracellular iron (II) concentrations in BMDMs differentiated under normoxia. We also analyzed the effect of ConA treatment on inflammatory gene expression in response to LPS stimulation by RT-PCR. ConA treatment significantly altered inflammatory gene expression in response to LPS (Fig. 8B) and reduced TET2 protein levels (Fig. 8C). Addition of iron to the culture medium partially restored intracellular iron (II) levels (Fig. 8A). Consistent with this partial restoration of iron (II), gene expression and TET2 protein levels were also partially restored (Fig. 8B, C). These results suggest that lysosomal inhibition by chronic hypoxia reduces the supply of ferrous iron, thereby suppressing LPS-induced TET2 accumulation and enhancing the expression of inflammatory genes.

[0032] [Effects of activated vitamin B6] We examined whether pyridoxal supplementation of CHyp-BMDMs restored lysosomal acidification (Figure 9A), Lamp1 protein expression (Figure 9B), and lysosomal-related gene (Hck, Tlr3) expression (Figure 9C). The results indicated that pyridoxal phosphate supplementation restored lysosomal acidification, Lamp1 protein expression, and lysosomal-related gene expression, suggesting that pyridoxal phosphate is sufficient to maintain lysosomal activity under chronic hypoxia. Furthermore, pyridoxal restored LPS-induced TET2 protein accumulation (Figure 9D) and suppressed IL-6 overproduction, as demonstrated by RT-PCR and ELISA (Figure 9E, F). These results suggest that chronic hypoxia induces a proinflammatory phenotype in macrophages by suppressing the activation of vitamin B6, which is required for lysosomal function. From the perspective of suppressing inflammatory responses, this suggests that supplementation with active vitamin B6 can suppress the expression of inflammatory cytokines and prevent the exacerbation of inflammation.

[0033] We investigated whether pyridoxal supplementation could have a similar effect in vivo. Mice exposed to hypoxia for 3 days were injected with LPS and Il6 induction in lung tissue was analyzed (Figure 9G). During hypoxia, pyridoxal was administered to mice using an implantable osmotic pump, which increased pyridoxal phosphate levels (Figure 9H, left). This resulted in the suppression of LPS-induced Il6 expression (Figure 9H, right). Therefore, pyridoxal supplementation can counteract the effects of chronic hypoxia in vivo.

[0034] As shown above, chronic hypoxia increases the expression of inflammatory cytokines and exacerbates inflammation. This is due to a pathway different from the previously shown HIF-mediated pathway, which is caused by a decrease in lysosomal activity, resulting in a decrease in the amount of intracellular pyridoxal phosphate synthesized in an oxygen-dependent manner (Figure 10). Active vitamin B6 can restore decreased lysosomal activity and alleviate the inflammatory response caused by chronic hypoxia.

Claims

1. A pharmaceutical composition for suppressing inflammation caused by chronic hypoxia, comprising: A pharmaceutical composition containing as an active ingredient a compound that enhances lysosomal function.

2. 2. The pharmaceutical composition according to claim 1, wherein the active ingredient is activated vitamin B6.

3. 3. The pharmaceutical composition according to claim 1, wherein the chronic hypoxic state is caused by a decrease in circulatory or respiratory function, or anemia.

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