Metabolic changes enhance necroptosis of type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis.

Previously, we found that Mycobacterium tuberculosis (Mtb) infection in type 2 diabetes mellitus (T2DM) mice enhances inflammatory cytokine production which drives pathological immune responses and mortality. In the current study, using a T2DM Mtb infection mice model, we determined the mechanisms that make T2DM mice alveolar macrophages (AMs) more inflammatory upon Mtb infection. Among various cell death pathways, necroptosis is a major pathway involved in inflammatory cytokine production by T2DM mice AMs. Anti-TNFR1 antibody treatment of Mtb-infected AMs from T2DM mice significantly reduced expression of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) (necroptosis markers) and IL-6 production. Metabolic profile comparison of Mtb-infected AMs from T2DM mice and Mtb-infected AMs of nondiabetic control mice indicated that 2-ketohexanoic acid and deoxyadenosine monophosphate were significantly abundant, and acetylcholine and pyridoxine (Vitamin B6) were significantly less abundant in T2DM mice AMs infected with Mtb. 2-Ketohexanoic acid enhanced expression of TNFR1, RIPK3, MLKL and inflammatory cytokine production in the lungs of Mtb-infected nondiabetic mice. In contrast, pyridoxine inhibited RIPK3, MLKL and enhanced expression of Caspase 3 (apoptosis marker) in the lungs of Mtb-infected T2DM mice. Our findings demonstrate that metabolic changes in Mtb-infected T2DM mice enhance TNFR1-mediated necroptosis of AMs, which leads to excess inflammation and lung pathology.

HIV-Differentiated Metabolite N-Acetyl-L-Alanine Dysregulates Human Natural Killer Cell Responses to Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) has latently infected over two billion people worldwide (LTBI) and caused ~1.6 million deaths in 2021. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10-20 times compared with HIV- LTBI+ patients. It is crucial to understand how HIV can dysregulate immune responses in LTBI+ individuals. Plasma samples collected from healthy and HIV-infected individuals were investigated using liquid chromatography-mass spectrometry (LC-MS), and the metabolic data were analyzed using the online platform Metabo-Analyst. ELISA, surface and intracellular staining, flow cytometry, and quantitative reverse-transcription PCR (qRT-PCR) were performed using standard procedures to determine the surface markers, cytokines, and other signaling molecule expressions. Seahorse extra-cellular flux assays were used to measure mitochondrial oxidative phosphorylation and glycolysis. Six metabolites were significantly less abundant, and two were significantly higher in abundance in HIV+ individuals compared with healthy donors. One of the HIV-upregulated metabolites, N-acetyl-L-alanine (ALA), inhibits pro-inflammatory cytokine IFN-γ production by the NK cells of LTBI+ individuals. ALA inhibits the glycolysis of LTBI+ individuals' NK cells in response to Mtb. Our findings demonstrate that HIV infection enhances plasma ALA levels to inhibit NK-cell-mediated immune responses to Mtb infection, offering a new understanding of the HIV-Mtb interaction and providing insights into the implication of nutrition intervention and therapy for HIV-Mtb co-infected patients.

HIV-differentiated metabolite N-Acetyl-L-Alanine dysregulates human natural killer cell responses to Mycobacterium tuberculosis infection.

Background: Mycobacterium tuberculosis ( Mtb ) has latently infected over two billion people worldwide (LTBI) and causes 1.8 million deaths each year. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10-20 times compared to the HIV-LTBI+ patients. It is crucial to understand how HIV can dysregulate immune responses in LTBI+ individuals. Methods: Plasma samples collected from healthy and HIV-infected individuals were investigated by liquid chromatography-mass spectrometry (LC-MS), and the metabolic data were analyzed using an online platform Metabo-Analyst. ELISA, surface and intracellular staining, flow cytometry, quantitative reverse transcription PCR (qRT-PCR) were performed by standard procedure to determine the surface markers, cytokines and other signaling molecule expression. Seahorse extra cellular flux assays were used to measure the mitochondrial oxidative phosphorylation and glycolysis. Results: Six metabolites were significantly less abundant, and two were significantly higher in abundance in HIV+ individuals compared to healthy donors. One of the HIV-upregulated metabolites, N-Acetyl-L-Alanine (ALA), inhibits pro-inflammatory cytokine IFN-□ production by NK cells of LTBI+ individuals. ALA inhibits glycolysis of LTBI+ individuals' NK cells in response to Mtb . Conclusions: Our findings demonstrate that HIV infection enhances plasma ALA levels to inhibit NK cell-mediated immune responses to Mtb infection, offering a new understanding of the HIV- Mtb interaction and providing the implication of nutrition intervention and therapy for HIV- Mtb co-infected patients.

Metabolites enhance innate resistance to human Mycobacterium tuberculosis infection.

To determine the mechanisms that mediate resistance to Mycobacterium tuberculosis (M. tuberculosis) infection in household contacts (HHCs) of patients with tuberculosis (TB), we followed 452 latent TB infection-negative (LTBI-) HHCs for 2 years. Those who remained LTBI- throughout the study were identified as nonconverters. At baseline, nonconverters had a higher percentage of CD14+ and CD3-CD56+CD27+CCR7+ memory-like natural killer (NK) cells. Using a whole-transcriptome and metabolomic approach, we identified deoxycorticosterone acetate as a metabolite with elevated concentrations in the plasma of nonconverters, and further studies showed that this metabolite enhanced glycolytic ATP flux in macrophages and restricted M. tuberculosis growth by enhancing antimicrobial peptide production through the expression of the surface receptor sialic acid binding Ig-like lectin-14. Another metabolite, 4-hydroxypyridine, from the plasma of nonconverters significantly enhanced the expansion of memory-like NK cells. Our findings demonstrate that increased levels of specific metabolites can regulate innate resistance against M. tuberculosis infection in HHCs of patients with TB who never develop LTBI or active TB.

Reduced thyroxine production in young household contacts of tuberculosis patients increases active tuberculosis disease risk.

In the current study, we followed 839 household contacts (HHCs) of tuberculosis (TB) patients for 2 years and identified the factors that enhanced the development of TB. Fourteen of the 17 HHCs who progressed to TB were in the 15- to 30-year-old age group. At baseline (the "0" time point, when all the individuals were healthy), the concentration of the thyroid hormone thyroxine (T4) was lower, and there were increased numbers of Tregs in PBMCs of TB progressors. At baseline, PBMCs from TB progressors stimulated with early secretory antigenic target 6 (ESAT-6) and 10 kDa culture filtrate antigen (CFP-10) produced less IL-1α. Thyroid hormones inhibited Mycobacterium tuberculosis (Mtb) growth in macrophages in an IL-1α-dependent manner. Mtb-infected Thra1PV/+ (mutant thyroid hormone receptor) mice had increased mortality and reduced IL-1α production. Our findings suggest that young HHCs who exhibit decreased production of thyroid hormones are at high risk of developing active TB disease.

Correction: IL-22 produced by type 3 innate lymphoid cells (ILC3s) reduces the mortality of type 2 diabetes mellitus (T2DM) mice infected with Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.ppat.1008140.].

Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection.

Macrophages are professional phagocytes known to play a vital role in controlling Mycobacterium tuberculosis (Mtb) infection and disease progression. Here we compare Mtb growth in mouse alveolar (AMs), peritoneal (PMs), and liver (Kupffer cells; KCs) macrophages and in bone marrow-derived monocytes (BDMs). KCs restrict Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections through enhanced autophagy. A metabolomics comparison of Mtb-infected macrophages indicates that ornithine and imidazole are two top-scoring metabolites in Mtb-infected KCs and that acetylcholine is the top-scoring in Mtb-infected AMs. Ornithine, imidazole and atropine (acetylcholine inhibitor) inhibit Mtb growth in AMs. Ornithine enhances AMPK mediated autophagy whereas imidazole directly kills Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or the two together restricts Mtb growth. Our study demonstrates that the metabolic differences between Mtb-infected AMs and KCs lead to differences in the restriction of Mtb growth.

BCG vaccination reduces the mortality of Mycobacterium tuberculosis-infected type 2 diabetes mellitus mice.

Diabetes is a significant risk factor for the development of active tuberculosis. In this study, we used a mouse model of type 2 diabetes mellitus (T2DM) to determine the effect of prior Bacillus Calmette-Guérin (BCG) vaccination on immune responses to Mycobacterium tuberculosis (Mtb) infection. We found that, at 6-7 months after Mtb infection, 90% of the Mtb-infected T2DM mice died, whereas only 50% of BCG-vaccinated T2DM-Mtb-infected mice died. Moreover, 40% of the PBS-treated uninfected T2DM mice and 30% of the uninfected BCG-vaccinated T2DM mice died, whereas all uninfected and infected nondiabetic mice survived. BCG vaccination was less effective in reducing the lung bacterial burden of Mtb-infected T2DM mice compared with Mtb-infected nondiabetic mice. BCG vaccination significantly reduced lung inflammation in Mtb-infected T2DM mice compared with that of unvaccinated T2DM mice infected with Mtb. Furthermore, reduced mortality of BCG-vaccinated Mtb-infected T2DM mice is associated with expansion of IL-13-producing CXCR3+ Tregs in the lungs of Mtb-infected T2DM mice. Recombinant IL-13 and Tregs from BCG-vaccinated Mtb-infected T2DM mice converted proinflammatory M1 macrophages to antiinflammatory M2 macrophages. Our findings suggest a potentially novel role for BCG in preventing excess inflammation and mortality in T2DM mice infected with Mtb.

IL-22 produced by type 3 innate lymphoid cells (ILC3s) reduces the mortality of type 2 diabetes mellitus (T2DM) mice infected with Mycobacterium tuberculosis.

Previously, we found that pathological immune responses enhance the mortality rate of Mycobacterium tuberculosis (Mtb)-infected mice with type 2 diabetes mellitus (T2DM). In the current study, we evaluated the role of the cytokine IL-22 (known to play a protective role in bacterial infections) and type 3 innate lymphoid cells (ILC3s) in regulating inflammation and mortality in Mtb-infected T2DM mice. IL-22 levels were significantly lower in Mtb-infected T2DM mice than in nondiabetic Mtb-infected mice. Similarly, serum IL-22 levels were significantly lower in tuberculosis (TB) patients with T2DM than in TB patients without T2DM. ILC3s were an important source of IL-22 in mice infected with Mtb, and recombinant IL-22 treatment or adoptive transfer of ILC3s prolonged the survival of Mtb-infected T2DM mice. Recombinant IL-22 treatment reduced serum insulin levels and improved lipid metabolism. Recombinant IL-22 treatment or ILC3 transfer prevented neutrophil accumulation near alveoli, inhibited neutrophil elastase 2 (ELA2) production and prevented epithelial cell damage, identifying a novel mechanism for IL-22 and ILC3-mediated inhibition of inflammation in T2DM mice infected with an intracellular pathogen. Our findings suggest that the IL-22 pathway may be a novel target for therapeutic intervention in T2DM patients with active TB disease.

Alcohol enhances type 1 interferon-α production and mortality in young mice infected with Mycobacterium tuberculosis.

In the current study, we used a mouse model and human blood samples to determine the effects of chronic alcohol consumption on immune responses during Mycobacterium tuberculosis (Mtb) infection. Alcohol increased the mortality of young mice but not old mice with Mtb infection. CD11b+Ly6G+ cells are the major source of IFN-α in the lungs of Mtb-infected alcohol-fed young mice, and IFN-α enhances macrophage necroptosis in the lungs. Treatment with an anti-IFNAR-1 antibody enhanced the survival of Mtb-infected alcohol-fed young mice. In response to Mtb, peripheral blood mononuclear cells (PBMCs) from alcoholic young healthy individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α than those from non-alcoholic young healthy LTBI+ individuals and alcoholic and non-alcoholic old healthy LTBI+ individuals. Our study demonstrates that alcohol enhances IFN-α production by CD11b+Ly6G+ cells in the lungs of young Mtb-infected mice, which leads to macrophage necroptosis and increased mortality. Our findings also suggest that young alcoholic LTBI+ individuals have a higher risk of developing active TB infection.

c-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong islet allograft survival in diabetic mice.

CD4+CD25+FoxP3+ cells (Tregs) inhibit inflammatory immune responses to allografts. Here, we found that co-transplantation of allogeneic pancreatic islets with Tregs that are defective in c-Jun N-terminal kinase 1 (JNK1) signaling prolongs islet allograft survival in the liver parenchyma of chemically induced diabetic mice (CDM). Adoptively transferred JNK1-/- but not wild-type (WT) Tregs survive longer in the liver parenchyma of CDM. JNK1-/- Tregs are resistant to apoptosis and express anti-apoptotic molecules. JNK1-/- Tregs express higher levels of lymphocyte activation gene-3 molecule (LAG-3) on their surface and produce higher amounts of the anti-inflammatory cytokine interleukin (IL)-10 compared with WT Tregs. JNK1-/- Tregs inhibit liver alloimmune responses more efficiently than WT Tregs. JNK1-/- but not WT Tregs are able to inhibit IL-17 and IL-21 production through enhanced LAG-3 expression and IL-10 production. Our study identifies a novel role of JNK1 signaling in Tregs that enhances islet allograft survival in the liver parenchyma of CDM.

Interleukin-21 Regulates Natural Killer Cell Responses During Mycobacterium tuberculosis Infection.

Background: In the current study, we determined the effects of interleukin (IL)-21 on human natural killer (NK) cells and monocyte responses during Mycobacterium tuberculosis (Mtb) infection. Methods: We found that Mtb stimulated CD4+ and NK T cells from healthy individuals with latent tuberculosis infection (LTBI+) are major sources of IL-21. CD4+ cells from tuberculosis patients secreted less IL-21 than did CD4+ cells from healthy LTBI+ individuals. Interleukin-21 had no direct effect on Mtb-stimulated monocytes. Results: Interleukin-21-activated NK cells produced interferon (IFN)-γ, perforin, granzyme B, and granulysin; lysed Mtb-infected monocytes; and reduced Mtb growth. Interleukin-21-activated NK cells also enhanced IL-1ß, IL-18, and CCL4/macrophage-inflammatory protein (MIP)-1ß production and reduced IL-10 production by Mtb-stimulated monocytes. Recombinant IL-21 (1) inhibited Mtb growth, (2) enhanced IFN-γ, IL-1ß, IL-18, and MIP-1ß, and (3) reduced IL-10 expression in the lungs of Mtb-infected Rag2 knockout mice. Conclusions: These findings suggest that activated T cells enhance NK cell responses to lyse Mtb-infected human monocytes and restrict Mtb growth in monocytes through IL-21 production. Interleukin-21-activated NK cells also enhance the immune response by augmenting IL-1ß, IL-18, and MIP-1ß production and reducing IL-10 production by monocytes in response to an intracellular pathogen.

IL-21 Receptor Signaling Is Essential for Optimal CD4+ T Cell Function and Control of Mycobacterium tuberculosis Infection in Mice.

In this study, we determined the role of IL-21R signaling in Mycobacterium tuberculosis infection, using IL-21R knockout (KO) mice. A total of 50% of M. tuberculosis H37Rv-infected IL-21R KO mice died in 6 mo compared with no deaths in infected wild type (WT) mice. M. tuberculosis-infected IL-21R KO mice had enhanced bacterial burden and reduced infiltration of Ag-specific T cells in lungs compared with M. tuberculosis-infected WT mice. Ag-specific T cells from the lungs of M. tuberculosis-infected IL-21R KO mice had increased expression of T cell inhibitory receptors, reduced expression of chemokine receptors, proliferated less, and produced less IFN- γ, compared with Ag-specific T cells from the lungs of M. tuberculosis-infected WT mice. T cells from M. tuberculosis-infected IL-21R KO mice were unable to induce optimal macrophage responses to M. tuberculosis. This may be due to a decrease in the Ag-specific T cell population. We also found that IL-21R signaling is associated with reduced expression of a transcriptional factor Eomesodermin and enhanced functional capacity of Ag-specific T cells of M. tuberculosis-infected mice. The sum of our findings suggests that IL-21R signaling is essential for the optimal control of M. tuberculosis infection.

A TLR9 agonist promotes IL-22-dependent pancreatic islet allograft survival in type 1 diabetic mice.

Pancreatic islet transplantation is a promising potential cure for type 1 diabetes (T1D). Islet allografts can survive long term in the liver parenchyma. Here we show that liver NK1.1+ cells induce allograft tolerance in a T1D mouse model. The tolerogenic effects of NK1.1+ cells are mediated through IL-22 production, which enhances allograft survival and increases insulin secretion. Increased expression of NKG2A by liver NK1.1+ cells in islet allograft-transplanted mice is involved in the production of IL-22 and in the reduced inflammatory response to allografts. Vaccination of T1D mice with a CpG oligonucleotide TLR9 agonist (ODN 1585) enhances expansion of IL-22-producing CD3-NK1.1+ cells in the liver and prolongs allograft survival. Our study identifies a role for liver NK1.1+ cells, IL-22 and CpG oligonucleotides in the induction of tolerance to islet allografts in the liver parenchyma.

NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection.

In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice.

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection.

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2-like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.

A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

In this study, we found that a subpopulation of CD4(+)CD25(+) (85% Foxp3(+)) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4(+)CD25(+) (85% Foxp3(+)) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+C)D25(+)Foxp3(+)D4GDI(+) cells do not produce IL-10, TGF-ß and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1ß, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+)) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+)CD25(+) (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

Phosphorylated STAT3 and PD-1 regulate IL-17 production and IL-23 receptor expression in Mycobacterium tuberculosis infection.

We studied the factors that regulate IL-23 receptor expression and IL-17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)-stimulated CD4(+) T cells from tuberculosis patients secreted less IL-17 than did CD4(+) T cells from healthy tuberculin reactors (PPD(+) ). M. tb-cultured monocytes from tuberculosis patients and PPD(+) donors expressed equal amounts of IL-23p19 mRNA and protein, suggesting that reduced IL-23 production is not responsible for decreased IL-17 production by tuberculosis patients. Freshly isolated and M. tb-stimulated CD4(+) T cells from tuberculosis patients had reduced IL-23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD(+) donors. STAT3 siRNA reduced IL-23 receptor expression and IL-17 production by CD4(+) T cells from PPD(+) donors. Tuberculosis patients had increased numbers of PD-1(+) T cells compared with healthy PPD(+) individuals. Anti-PD-1 antibody enhanced pSTAT3 and IL-23R expression and IL-17 production by M. tb-cultured CD4(+) T cells of tuberculosis patients. Anti-tuberculosis therapy decreased PD-1 expression, increased IL-17 and IFN-γ production and pSTAT3 and IL-23R expression. These findings demonstrate that increased PD-1 expression and decreased pSTAT3 expression reduce IL-23 receptor expression and IL-17 production by CD4(+) T cells of tuberculosis patients.

Interleukin 22 inhibits intracellular growth of Mycobacterium tuberculosis by enhancing calgranulin A expression.

Previously, we found that interleukin 22 (IL-22) inhibits intracellular growth of Mycobacterium tuberculosis in human monocyte-derived macrophages (MDMs). In the current study, we determined the mechanisms underlying these effects. We found that W7, a phagolysosomal fusion inhibitor, abrogates IL-22-dependent M. tuberculosis growth inhibition in MDMs, suggesting that IL-22 acts through enhanced phagolysosomal fusion. Our microarray analysis indicated that recombinant IL-22 (rIL-22) enhances the expression of an intracellular signaling molecule, calgranulin A. This was confirmed by real-time polymerase chain reaction, Western blot, and confocal microscopy. Calgranulin A small interfering RNA (siRNA) abrogated rIL-22-dependent growth inhibition of M. tuberculosis in MDMs. IL-22 enhanced Rab7 expression and downregulated Rab14 expression of M. tuberculosis-infected MDMs, and these effects were reversed by calgranulin A siRNA. These results suggest that M. tuberculosis growth inhibition by IL-22 depends on calgranulin A and enhanced phagolysosomal fusion, which is associated with increased Rab7 and reduced Rab14 expression.