Cytokine/Chemokine Release Patterns and Transcriptomic Profiles of LPS/IFNγ-Activated Human Macrophages Differentiated with Heat-Killed Mycobacterium obuense, M-CSF, or GM-CSF.

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment.

Mitochondrial bioenergetics, uncoupling protein-2 activity, and reactive oxygen species production in the small intestine of a TNBS-induced colitis rat model.

Inflammatory bowel disease (IBD) is often associated with a decrease in energy-dependent nutrient uptake across the jejunum that serves as the main site for absorption in the small intestine. This association has prompted us to investigate the bioenergetics underlying the alterations in jejunal absorption in 2,4,6-trinitrobenzenesulfonic acid-induced colitis in rats. We have found that mitochondrial oxygen consumption did not change in state 2 and state 3 respirations but showed an increase in state 4 respiration indicating a decrease in the respiratory control ratio of jejunal mitochondria during the peak of inflammation. This decrease in the coupling state was found to be guanosine diphosphate-sensitive, hence, implicating the involvement of uncoupling protein-2 (UCP2). Furthermore, the study has reported that the production of reactive oxygen species (ROS), known to be activators of UCP2, correlated negatively with UCP2 activity. Thus, we suggest that ROS production in the jejunum might be activating UCP2 which has an antioxidant activity, and that uncoupling of the mitochondria decreases the efficiency of energy production, leading to a decrease in energy-dependent nutrient absorption. Hence, this study is the first to account for an involvement of energy production and a role for UCP2 in the absorptive abnormalities of the small intestine in animal models of colitis.

Absence of effect of the antiretrovirals Duovir and Viraday on mitochondrial bioenergetics.

Most toxicity associated with antiretroviral drugs is thought to result from disruption of mitochondrial function. Unfortunately, there are no validated laboratory markers for clinically assessing the onset of mitochondrial toxicity associated with antiretroviral therapy. In a previous study on mitochondrial hepatocytes, the protease inhibitor lopimune was shown to induce mitochondrial toxicity by increasing reactive oxygen species (ROS) production and decreasing respiratory control ratio (RCR) reflecting compromised mitochondrial efficiency in adenosine triphosphate production. Mitochondrial dysfunction and ROS production were directly correlated with the expression of uncoupling protein 2 (UCP2). In the current study we aim to determine the toxicity of nucleoside or nucleotide and nonnucleoside reverse-transcriptase inhibitors, Duovir and Viraday on liver mitochondria isolated from treated mice by monitoring UCP2 expression. Our results showed that both Duovir and Viraday had no effect on mitochondrial respiration states 2, 3, 4, and on RCR. In addition, ROS generation and UCP2 expression were not affected. In conclusion, our results indicate the difference in the mechanism of action of distinct classes of antiretroviral drugs on mitochondrial functions and may associate UCP2 expression with subclinical mitochondrial damage as marker of cellular oxidative stress.

Defining Genome-Wide Expression and Phenotypic Contextual Cues in Macrophages Generated by Granulocyte/Macrophage Colony-Stimulating Factor, Macrophage Colony-Stimulating Factor, and Heat-Killed Mycobacteria.

Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecular underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefined. To fill this void, we sought to perform immunophenotyping, chemokine/cytokine release analysis and genome-wide characterization of monocyte-derived macrophages (MDM) in which monocytes were originally isolated from healthy donors and differentiated by HK M. obuense (Mob-MDM) relative to macrophage colony-stimulating factor (M-MDM) and granulocyte/macrophage colony-stimulating factor (GM-MDM). Immunophenotyping and cytokine release analysis revealed downregulated surface expression of CD36, decreased spontaneous release of CCL2 and increased spontaneous secretion of CCL5, CXCL8/IL-8, IL-6, and TNF-α in Mob-MDM relative to M-MDM and GM-MDM. Analysis of cytostatic activity showed that Mob-MDM exhibited similar growth inhibitory effects on immortalized and malignant epithelial cells compared with GM-MDM but at an elevated rate relative to M-MDM. To understand global cues in Mob-MDM, we performed comparative RNA-sequencing (RNA-Seq) analysis of Mob-MDM relative to GM-MDM and M-MDM (n = 4 donors). Clustering analysis underscored expression profiles (n = 256) that were significantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/cytokines and their receptors, enzymes and transcriptions factors. Topological functional analysis of these profiles identified pathways and gene sets linked to Mob-MDM phenotype including nitric oxide production, acute phase response signaling and microbe recognition pathways as well as signaling cues mediated by the proinflammatory cytokine, interferon-gamma, and the intracellular pattern recognition receptor, nucleotide-binding oligomerization domain-containing protein 2. Taken together, our study highlights molecular immune phenotypes and global signaling cues in Mob-MDM that may underlie immunomodulatory properties of HK M. obuense. Such properties could be of valuable use in immunotherapy approaches such as adoptive cell therapy against cancer.

Immunomodulatory effects of heat-killed Mycobacterium obuense on human blood dendritic cells.

Heat-killed (HK) Mycobacterium obuense is a novel immunomodulator, currently undergoing clinical evaluation as an immunotherapeutic agent in the treatment of cancer. Here, we examined the effect of in vitro exposure to HK M. obuense on the expression of different categories of surface receptors on human blood myeloid (m) and plasmacytoid (p) DCs. Moreover, we have characterized the cytokine and chemokine secretion patterns of purified total blood DCs stimulated with HK M. obuense. HK M. obuense significantly up-regulated the expression of CD11c, CD80, CD83, CD86, CD274 and MHC class II in whole-blood mDCs and CD80, CD123 and MHC class II in whole-blood pDCs. Down-regulation of CD195 expression in both DC subpopulations was also noted. Further analysis showed that HK M. obuense up-regulated the expression of CD80, CD83 and MHC class II on purified blood DC subpopulations. TLR2 and TLR1 were also identified to be engaged in mediating the HK M. obuense-induced up-regulation of surface receptor expression on whole blood mDCs. In addition, our data demonstrated that HK M. obuense augmented the secretion of CCL4, CCL5, CCL22, CXCL8, IL-6, IL-12p40 and TNF-α by purified total blood DCs. Taken together, our data suggest that HK M. obuense exerts potent differential immunomodulatory effects on human DC subpopulations.

Differential regulation of surface receptor expression, proliferation, and apoptosis in HaCaT cells stimulated with interferon-γ, interleukin-4, tumor necrosis factor-α, or muramyl dipeptide.

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively. A significant decrease in the expression of CD49d was found upon treatment with interleukin-4. Interferon gamma and tumor necrosis factor alpha increased the expression of intercellular adhesion molecule 1 and major histocompatibility complex class I, whereas major histocompatibility complex class II and CD1b were only upregulated by interferon gamma. Interferon gamma and tumor necrosis factor alpha had opposite effects regarding CD119 expression, with the former downregulating, while the latter upregulating its expression. Of the stimuli tested, only interferon gamma and tumor necrosis factor alpha significantly inhibited proliferation of HaCaT cells, yet only interferon gamma played a significant role in inducing HaCaT cell apoptosis. Our data demonstrate differential effects of the three tested cytokines on keratinocytes and reveal that the absence of HaCaT cell responses to muramyl dipeptide is associated with undetectable levels of its cytoplasmic receptor, nucleotide-binding oligomerization domain-containing protein 2.

Analysis of the immunomodulatory properties of two heat-killed mycobacterial preparations in a human whole blood model.

The significant role played by mycobacteria in modulating immune responses through enhancing the crosstalk between innate and adaptive immunity has been highlighted in several studies. Owing to their unique antigenic profile, heat killed (HK) preparations of rapid-growing mycobacteria, currently undergoing clinical development, have been assessed as adjuvant therapy in various diseases. The purpose of this study is to investigate the regulation of leukocyte surface receptors, in whole blood from healthy donors, following in vitro stimulation with HK Mycobacterium vaccae (M. vaccae) or M. obuense. We have demonstrated the ability of both mycobacterial preparations to target monocytes and neutrophils and to regulate the surface expression of selected adhesion receptors, antigen-presenting and costimulatory receptors, pattern recognition receptors, complement and Fc receptors, as well as cytokine/chemokine receptors. Toll-like receptors (TLRs) 1 and 2 were also shown to be involved in mediating the M. obuense-induced upregulation of selected surface receptors on monocytes. Whole blood stimulation with M. vaccae or M. obuense resulted in a significant increase in the secretion of a specific set of cytokines and chemokines. Both mycobacterial preparations induced strong antigen-specific proliferative responses in peripheral blood mononuclear cells. Collectively, our data shows that M. vaccae and M. obuense have the potential to act as potent immunomodulators. Future research based on these findings may reveal novel immune pathways induced by these preparations with potential implication for their use in diverse immunotherapeutic approaches.

Lopimune-induced mitochondrial toxicity is attenuated by increased uncoupling protein-2 level in treated mouse hepatocytes.

Although the protease inhibitor (PI) Lopimune has proven to be effective, no studies have examined the side effects of Lopimune on mitochondrial bioenergetics in hepatocytes. The objective of the present study is to evaluate mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2) in mouse hepatocytes following Lopimune administration. Mitochondria were extracted from mouse liver using differential centrifugation and hepatocytes were isolated by the collagenase perfusion procedure. Mitochondrial respiration was measured using a Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometry using a 2',7'-dichlorofluorescin diacetate probe and UCP2 protein expression was detected by Western blotting. We found that Lopimune induced a significant decrease of approximately 30% in the respiratory control ratio (RCR) starting from day 4 until day 9 of treatment. This decrease was due to an increase in state 4 respiration, reflecting an increase in mitochondrial proton leak. State 2 and state 3 respirations were not affected. Moreover, ROS production significantly increased by about 2-fold after day 1 of treatment and decreased after day 3, returning to the resting level on day 5. Interestingly, UCP2 which is absent from control hepatocytes, was expressed starting from day 4 of treatment. Our findings indicate that Lopimune-induced proton leak, mediated by UCP2, may represent a response to inhibit the production of ROS as a negative feedback regulatory mechanism. These results imply a potential involvement of UCP2 in the regulation of oxidative stress and add new insights into the understanding of mitochondrial toxicity induced by PIs.

Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein 2 and the production of reactive oxygen and reactive nitrogen species.

The synthetic immunomodulator muramyl dipeptide (MDP) has been shown to induce, in vivo, mitochondrial proton leak. In the present work, we extended these findings to the cellular level and confirmed the effects of MDP in vitro on murine macrophages. The macrophage system was then used to analyse the mechanism of the MDP-induced mitochondrial proton leak. Our results demonstrate that the cellular levels of superoxide anion and nitric oxide were significantly elevated in response to MDP. Moreover, isolated mitochondria from cells treated with MDP presented a significant decrease in respiratory control ratio, an effect that was absent following treatment with a non-toxic analogue such as murabutide. Stimulation of cells with MDP, but not with murabutide, rapidly upregulates the expression of the mitochondrial protein uncoupling protein 2 (UCP2), and pretreatment with vitamin E attenuates upregulation of UCP2. These findings suggest that the MDP-induced reactive species upregulate UCP2 expression in order to counteract the effects of MDP on mitochondrial respiratory efficiency.

The common mycobacterial antigens and their importance in the treatment of disease.

The mycobacteria are one of a number of genera making up the aerobic Actinomycetales. Their antigens demonstrable by immuno-precipitation methods can be divided into four groups. The group i antigens, common to all mycobacterial species, cross-react with their counterparts in animal cells, largely derived from mitochondria. Notable amongst these antigens are the heat-shock, or stress, proteins and possibly bacterial sugars. Tests of cell-mediated immunity show that people can be separated by their responsiveness in skin-test, or lymphocyte proliferation techniques, into four categories of responders. Category 1 individuals respond to all mycobacterial reagents through recognition of the group i antigens. Many chronic diseases are associated with a lack of cell-mediated responsiveness to the group i antigens, and have a raised antibody titre to them. This reflects a predominance of T helper 2 activity and reduced T helper 1 responsiveness as part of the pathogenesis of their diseases, which include chronic bacterial, viral and parasitic infections, allergies, auto-immunities and neoplasms. Packaged together, the group i antigens and the cell-wall adjuvants of selected aerobic Actinomycetales make potent immuno-modulatory reagents. An example is heat-killed Mycobacterium vaccae, useful in both prevention and treatment of disease. Treatment with such reagents results in alleviation of disease, restoration of cellular responsiveness to the common mycobacterial antigens and a decrease in antibody titres to them. This new approach to treatment for such a wide range of diseases has few disadvantageous side effects and can accompany other non-immunosuppressive therapies.