The Link Between Dysregulated Immunometabolism and Vascular Damage: Implications for the Development of Atherosclerosis in Systemic Lupus Erythematosus and Other Rheumatic Diseases.

A bimodal pattern of mortality in systemic lupus erythematosus (SLE) exists. Early-stage deaths are predominantly caused by infection, whereas later-stage deaths are mainly caused by atherosclerotic disease. Further, although SLE-related mortality has reduced considerably in recent years, cardiovascular (CV) events remain one of the leading causes of death in people with SLE. Accelerated atherosclerosis in SLE is attributed to both an increase in traditional CV risk factors and the inflammatory effects of SLE itself. Many of these changes occur within the microenvironment of the vascular-immune interface, the site of atherosclerotic plaque development. Here, an intimate interaction between endothelial cells, vascular smooth muscle cells, and immune cells dictates physiological vs pathological responses to a chronic type 1 interferon environment. Low-density neutrophils (LDNs) have also been implicated in eliciting vasculature-damaging effects at such lesion sites. These changes are thought to be governed by dysfunctional metabolism of immune cells in this niche due at least in part to the chronic induction of type 1 interferons. Understanding these novel pathophysiological mechanisms and metabolic pathways may unveil potential innovative pharmacological targets and therapeutic opportunities for atherosclerosis, as well as shed light on the development of premature atherosclerosis in patients with SLE who develop CV events.

Chronic IFNα treatment induces leukopoiesis, increased plasma succinate and immune cell metabolic rewiring.

Although clinically effective, the actions of IFNα, either produced endogenously or by therapeutic delivery, remain poorly understood. Emblematic of this research gap is the disparate array of notable side effects that occur in susceptible individuals, such as neuropsychiatric consequences, autoimmune phenomena, and infectious complications. We hypothesised that these complications are driven at least in part by dysregulated cellular metabolism. Male Wistar rats were treated with either 170,000 IU/kg human recombinant IFNα-2a or BSA/saline (0.9% NaCl) three times per week for three weeks. Bone marrow (BM) immune cells were isolated from the excised femurs for glycolytic rate and mitochondrial function assessment using Agilent Seahorse Technology. Frequencies of immune cell populations were assessed by flow cytometry to determine whether leukopoietic changes had occurred in both blood and BM. Plasma levels of lactate and succinate were also determined. BMDMs were metabolically assessed as above, as well as their metabolic response to an antigenic stimulus (iH37Rv). We observed that BM immune cells from IFN-treated rats exhibit a hypermetabolic state (increased basal OCR/GlycoPER) with decreased mitochondrial metabolic respiration and increased non-mitochondrial OCR. Flow cytometry results indicated an increase in immature granulocytes (RP1- SSChi CD45lo) only in the blood, together with increased succinate levels in the plasma. BMDMs from IFN-treated rats retained the hypermetabolic phenotype after differentiation and failed to induce a step-up in glycolysis and mitochondrial respiration after bacterial stimulation. This work provides the first evidence of the effects of IFNα treatment in inducing hypermetabolic immune features that are associated with markers of inflammation, leukopoiesis, and defective responses to bacterial stimulation.

Mitochondrial bioenergetic changes in systemic lupus erythematosus immune cell subsets: Contributions to pathogenesis and clinical applications.

The association of dysregulated metabolism in systemic lupus erythematosus (SLE) pathogenesis has prompted investigations into metabolic rewiring and the involvement of mitochondrial metabolism as a driver of disease through NLRP3 inflammasome activation, disruption of mitochondrial DNA maintenance, and pro-inflammatory cytokine release. The use of Agilent Seahorse Technology to gain functional in situ metabolic insights of selected cell types from SLE patients has identified key parameters that are dysregulated during disease. Mitochondrial functional assessments specifically can detect dysfunction through oxygen consumption rate (OCR), spare respiratory capacity, and maximal respiration measurements, which, when coupled with disease activity scores could show potential as markers of disease activity. CD4+ and CD8 + T cells have been assessed in this way and show that oxygen consumption rate, spare respiratory capacity, and maximal respiration are blunted in CD8 + T cells, with results not being as clear cut in CD4 + T cells. Additionally, glutamine, processed by mitochondrial substrate level phosphorylation is emerging as a key role player in the expansion and differentiation of Th1, Th17, ϒδ T cells, and plasmablasts. The role that circulating leukocytes play in acting as bioenergetic biomarkers of diseases such as diabetes suggests that this may also be a tool to detect preclinical SLE. Therefore, the metabolic characterization of immune cell subsets and the collection of metabolic data during interventions is also essential. The delineation of the metabolic tuning of immune cells in this way could lead to novel strategies in treating metabolically demanding processes characteristic of autoimmune diseases such as SLE.

Lactate Alters Metabolism in Human Macrophages and Improves Their Ability to Kill Mycobacterium tuberculosis.

In order to mount an appropriate immune response to infection, the macrophage must alter its metabolism by increasing aerobic glycolysis and concomitantly decreasing oxidative phosphorylation; a process known as the Warburg effect. Consequently, lactate, the end-product of glycolysis, accumulates in the extracellular environment. The subsequent effect of lactate on surrounding macrophages is poorly understood. Mycobacterium tuberculosis (Mtb), the causative organism of Tuberculosis (TB), is phagocytosed by macrophages in the airways. Mtb infected macrophages upregulate aerobic glycolysis and effector functions to try to kill the bacteria. Our lab has previously shown that human macrophages produce lactate in response to infection with Mtb. Although lactate has largely been considered a waste product of aerobic glycolysis, we hypothesised that the presence of extracellular lactate would impact subsequent immunometabolic responses and modulate macrophage function. We demonstrate that the presence of exogenous lactate has an immediate effect on the cellular metabolism of resting human macrophages; causing a decrease in extracellular acidification rate (ECAR; analogous to the rate of glycolysis) and an increase in the oxygen consumption rate (OCR; analogous to oxidative phosphorylation). When lactate-treated macrophages were stimulated with Mtb or LPS, glycolysis proceeds to increase immediately upon stimulation but oxidative phosphorylation remains stable compared with untreated cells that display a decrease in OCR. This resulted in a significantly reduced ECAR/OCR ratio early in response to stimulation. Since altered metabolism is intrinsically linked to macrophage function, we examined the effect of lactate on macrophage cytokine production and ability to kill Mtb. Lactate significantly reduced the concentrations of TNF and IL-1ß produced by human macrophages in response to Mtb but did not alter IL-10 and IL-6 production. In addition, lactate significantly improved bacillary clearance in human macrophages infected with Mtb, through a mechanism that is, at least in part, mediated by promoting autophagy. These data indicate that lactate, the product of glycolysis, has a negative feedback effect on macrophages resulting in an attenuated glycolytic shift upon subsequent stimulation and reduced pro-inflammatory cytokine production. Interestingly, this pro-resolution effect of lactate is associated with increased capacity to kill Mtb.

Immunoglobulin profile and B-cell frequencies are altered with changes in the cellular microenvironment independent of the stimulation conditions.

INTRODUCTION: B-cells are essential in the defense against Mycobacterium tuberculosis. Studies on isolated cells may not accurately reflect the responses that occur in vivo due to the presence of other cells. This study elucidated the influence of microenvironment complexity on B-cell polarization and function in the context of tuberculosis disease. METHODS: B-cell function was tested in whole blood, peripheral blood mononuclear cells (PBMCs), and as isolated cells. The different fractions were stimulated and the B-cell phenotype and immunoglobulin profiles analyzed. RESULTS: The immunoglobulin profile and developmental B-cell frequencies varied for each of the investigated sample types, while in an isolated cellular environment, secretion of immunoglobulin isotypes immunoglobulin A (IgA), IgG2, and IgG3 was hampered. The differences in the immunoglobulin profile highlight the importance of cell-cell communication for B-cell activation. Furthermore, a decrease in marginal zone B-cell frequencies and an increase in T1 B-cells was observed following cell isolation, indicating impaired B-cell development in response to in vitro antigenic stimulation in isolation. CONCLUSION: Our results suggest that humoral B-cell function and development was impaired likely due to a lack of costimulatory signals from other cell types. Thus, B-cell function should ideally be studied in a PBMC or whole blood fraction.

Distinct host-immune response toward species related intracellular mycobacterial killing: A transcriptomic study.

The comparison of the host immune response when challenged with pathogenic and nonpathogenic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.

Tween 80 induces a carbon flux rerouting in Mycobacterium tuberculosis.

As a means to increase the growth rate and reduce aggregation, Tween 80 is routinely added to growth media during mycobacterial culturing. This detergent has, however, been associated with causing alterations to the morphology, pathogenicity and virulence of these bacteria. In an attempt to better understand the underlying mechanism of these alterations, we investigated the effect of Tween 80 on the metabolomes of a M. tuberculosis lab strain (H37Rv) and multidrug-resistant clinical strain (R179), using GC-GCxTOF-MS metabolomics. The metabolite markers identified indicated Tween 80-induced disparities in the central carbon metabolism of both strains, with an upregulation in the glyoxylate cycle, glucogenogenesis and the pentose phosphate pathway. The results also signified an increased production of mycobacterial biosynthetic precursors such as triacylglycerols, proteinogenic amino acids and nucleotide precursors, in the presence of the detergent. Collectively, these metabolome variations mimic the phenotypic changes observed when M. tuberculosis is grown in vivo, in a lipid rich environment. However, in addition to the increased availability of oleic acid as a carbon source from Tween 80, the observed variations, and the morphological changes associated with the detergent, could also be a result of an overall stress response in these bacteria. This study is the first to identify specific metabolome variations related to the addition of Tween 80 to the growth media during M. tuberculosis culturing. The consideration of these results during the method development and data interpretation phases of future metabolomics investigations will improve the quality of the analyses as well as the credibility of potential research outcomes. These results will also assist in the interpretation of research questions specifically aimed at aspects of mycobacterial metabolism, even when using other methodologies such as transcriptomics or fluxomics.

PI3-Kinase δγ Catalytic Isoforms Regulate the Th-17 Response in Tuberculosis.

Although IL17A plays a protective role at the mucosal surface, when IL17A signaling becomes dysregulated, a pathological response is locally induced. At the early stages of Mycobacterium tuberculosis (M.tb) infection, IL17A contributes to granuloma formation and pathogen containment. In contrast, during disease progression, a dysregulated IL17A hyperinflammatory response drives tissue destruction through enhanced neutrophil recruitment. Cumulative research has implicated the PI3-Kinase pathways as one of the most relevant in the pathophysiology of inflammation. Evidence shows that IL-17A secretion and the expansion of the Th17 population is dependant in PI3-Kinase signaling, with the p110δ and p110γ isoforms playing a prominent role. The p110γ isoform promotes disease progression through dampening of the Th17 response, preventing pathogen clearance and containment. The p110γ gene, PIK3CG is downregulated in TB patients during late-stage disease when compared to healthy controls, demonstrating an important modulatory role for this isoform during TB. Conversely, the p110δ isoform induces IL-17A release from pulmonary γδ T-cells, committed Th17 cells and promotes neutrophil recruitment to the lung. Inhibiting this isoform not only suppresses IL-17A secretion from Th17 cells, but it also inhibits cytokine production from multiple T-helper cell types. Since increased IL-17A levels are observed to be localized in the lung compartments (BAL and lymphocytes) in comparison to circulating levels, an inhalable PI3Kδ inhibitor, which is currently utilized for inflammatory airway diseases characterized by IL-17A over-secretion, may be a therapeutic option for active TB disease.

OAS1, OAS2 and OAS3 restrict intracellular M. tb replication and enhance cytokine secretion.

The 2',5' (OASs) are known as mediators of the antiviral response system through activation of the RNA cleavage pathway. Interestingly, we observe OAS1, OAS2 and OAS3 upregulation in a number of gene expression signatures which discriminate active TB from latent TB infection, however their biological role during bacterial infection has not yet been elucidated. We observed that the expression of these genes was associated with pathogenicity and virulence of mycobacteria as infection with Mycobacterium bovis BCG failed to significantly induce OAS expression. Further, we observed that after silencing of these genes, M. tb CFU counts increased significantly 96h post-infection in comparison to the respective controls. Luminex revealed that OAS silencing significantly decreased IL-1ß, TNF-α and MCP-1 and had no effect of IL-10 secretion. We show for the first time that OAS1, 2 and 3 restrict intracellular pathogenic mycobacterial replication and enhance pro-inflammatory cytokine secretion.

Susceptibility to Tuberculosis Is Associated With PI3K-Dependent Increased Mobilization of Neutrophils.

Neutrophilia is a condition commonly observed in patients with late-stage tuberculosis, but evidence suggests that increased neutrophil influx begins early after infection in susceptible hosts and functions to promote a nutrient-replete niche that promotes Mycobacterium tuberculosis survival and persistence. As the disease progresses, an increase in the number of neutrophil-like cells is observed, all of which exhibit characteristics associated with (i) phenotypic and biochemical features of immaturity, (ii) the inability to activate T-cells, (iii) hyper-inflammation, and (iv) prolonged survival. Transcriptomics reveal a common set of molecules associated with the PI3-Kinase pathway that are dysregulated in patients with active tuberculosis. Closer inspection of their individual biological roles reveal their ability to modulate the IL-17/G-CSF axis, induce leukocyte receptor activation, and regulate apoptosis and motility. This review draws attention to neutrophil hyper-reactivity as a driving force for both the establishment and progression of tuberculosis disease in susceptible individuals.

OAS1, 2, and 3: Significance During Active Tuberculosis?

Evidence to-date points to a detrimental role of the type I IFNs during tuberculosis. The mechanisms underpinning the IFNαß-mediated exacerbation of the disease is unclear. The 2'-5'-oligoadenylate synthetases (OAS), namely OAS1, OAS2 and OAS3 are part of the interferon-induced genes which until now have been synonymous with an anti-viral function. Blood transcriptome profiling has continuously observed their upregulation in a number of gene expression signatures which discriminate active TB from latent TB infection, however the role of the OASs and the effect that their expression has on the pathogenesis and persistence of TB is unknown. Evidence suggests that the OASs exhibit other cellular functions which include the induction of apoptosis, enhancement of IFNαß signalling, immune cell receptor modulation and autophagy. We propose that i) during the late stages of disease, sustained RNaseL expression through OAS activation enhances type I IFN signalling and, ii) that they may exhibit immune-modulatory capabilities.

The Association of OASL and Type I Interferons in the Pathogenesis and Survival of Intracellular Replicating Bacterial Species.

The type I IFN response quickly became associated with its role in the innate immune response to viral infection. The past few years have seen the significance of IFNs expand in breadth to include non-viral pathogens. Previous work has identified that following viral infection, type I IFN signaling induces the production of the 2'-5'-oligoadenylate synthetase (OAS) family, which include OAS1, OAS2, OAS3, and OAS-like (OASL) protein. OASL was identified to be strongly induced following viral infection through engaging the RNA sensor RIG-I and increasing signaling through this pathway to enhance the anti-viral type I IFN response. Surprisingly, infection with viral dsDNA revealed an IFN inhibitory role and therefore pro-viral function of OASL through the inhibition of the cGAS cytosolic DNA sensing mechanism. Intracellular bacteria are able to activate the cytosolic DNA sensing pathway, however the role of OASL during bacterial infection is largely unknown. Vacuolar pathogenic microbes such as mycobacteria induce OASL early post infection, where it functions in a prosurvival fashion by inhibiting autophagic mechanisms and antimicrobial peptide expression. This suggests an underestimated role of OASL in the innate immune response to infection with a variety of pathogens and points to OASL-associated modulation of the type I IFN response. OASL may therefore play a critical role in defining the outcome of infection. We provide a brief update on the recent developments of the OAS family of proteins in response to DNA and RNA virus infections, as well as discuss evidence of Oasl expression in response to a number of cytosolic and vacuolar replicating bacterial pathogens.

RNAseq reveals hypervirulence-specific host responses to M. tuberculosis infection.

The distinguishing factors that characterize the host response to infection with virulent Mycobacterium tuberculosis (M.tb) are largely confounding. We present an infection study with 2 genetically closely related M.tb strains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) Beijing M. tuberculosis clinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these 2 transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed 2 early-response genes (ier3 and saa3) and 2 host-defense genes (oasl1 and slpi) that were significantly upregulated by the hypervirulent strain. The role of these genes under M.tb infection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role during infection with virulent M.tb is required.

The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach.

During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.

Bcl-2 confers survival in cisplatin treated cervical cancer cells: circumventing cisplatin dose-dependent toxicity and resistance.

BACKGROUND: Cisplatin is the main chemotherapeutic drug for the treatment of cervical cancers, however resistance to cisplatin is increasingly common and therefore has limited the efficacy and use of this drug in the clinic. Dose-dependent toxicity poses an additional challenge since patients suffer long-term and often permanent side-effects after treatment. Bcl-2 up-regulation has been implicated in the resistance to cisplatin in a variety of cancer cell lines, however its role in cervical cancer is confounding. METHODS: A low, non-cytotoxic concentration of cisplatin was used in the treatment of HeLa and CaSki cells. Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin. To assess the reliance of the cervical cancer cells on Bcl-2 in the presence of cisplatin, Bcl-2 knock-down was achieved through RNA interference, where after apoptosis was assessed through PARP cleavage (Western blotting), Caspase activity (Caspase-Glo(©)) and PI inclusion analysis (Flow cytometry). Finally, pre-malignant and malignant cervical tissue was analysed for the presence of Bcl-2 through Western blotting and immunofluorescence. RESULTS: Cervical cancer cells upregulate Bcl-2 when treated with a non-cytotoxic concentration of cisplatin, which when silenced, effectively enhanced cisplatin sensitivity, and therefore significantly induced apoptosis. Analysis of the expression profile of Bcl-2 in cervical tissue revealed its up-regulation in cervical carcinoma, which agrees with results obtained from the in vitro data. CONCLUSIONS: Our data strongly suggest that utilising a lower dose of cisplatin is feasible when combined with Bcl-2 silencing as an adjuvant treatment, thereby improving both the dose-dependent toxicity, as well as cervical cancer resistance.

A nontoxic concentration of cisplatin induces autophagy in cervical cancer: selective cancer cell death with autophagy inhibition as an adjuvant treatment.

BACKGROUND: Increasing resistance to cisplatin as well as the severity of the adverse effects limit the use of this drug, particularly at high doses. Evidence has implicated the importance of autophagy in cancer resistance as well as the fact that various chemotherapy agents induce autophagy in cancer cells. We therefore aimed to first assess the role of autophagy in cisplatin treatment and second to assess whether a nontoxic concentration of cisplatin, together with autophagy inhibition, is able to maintain its cancer-specific cytotoxic action. METHODS: Three human cervical cell lines were used: a noncancerous ectocervical epithelial cell line (Ect1/E6E7) and 2 cancerous cervical cell lines (HeLa and CaSki). Autophagy was monitored through the presence of the classical protein markers LC-3 II and p62 under basal and treatment conditions, and inhibited using bafilomycin and autophagy protein 5 (ATG5) siRNA under treatment conditions. Cell death was analyzed through examination of the apoptotic markers PARP and caspase-3 through Western blotting, as well as the Caspase-Glo assay to confirm caspase-3/7 activity. Cervical biopsies were analyzed for the presence of LC-3 using Western blotting and immunofluorescence to determine if a correlation between autophagic levels and the progression of the disease exists. RESULTS: Cervical cancer cells exhibit increased basal autophagic levels in comparison to the noncancerous counterparts. Cisplatin treatment enhanced autophagic activity in all 3 cell lines. Inhibition of this autophagic response together with cisplatin treatment leads to significant increases in cancer cell death. Expression profiles of LC-3 in normal, premalignant (low-grade and high-grade squamous intraepithelial lesion), and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma cervical tissue, which emphasized the role of autophagy in the progression of the disease. CONCLUSIONS: The inhibition of autophagy improves the cytotoxicity of a nontoxic concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer.