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ABSTRACT Hypoxia-inducible factor 1α (HIF-1α) regulates the immunometabolic phenotype of macrophages, including the orchestration of inflammatory and antimicrobial processes. Macrophages deficient in HIF-1α produce excessive quantities of the anti-inflammatory cytokine interleukin 10 (IL-10) during infection with the intracellular fungal pathogen Histoplasma capsulatum (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565–579, 2016, https://doi.org/10.4049/jimmunol.1600342). Thus, the macrophage fails to become activated in response to proinflammatory cytokines and remains the intracellular niche of the pathogen. Here, we identify the tricarboxylic acid (TCA) cycle metabolite fumarate as the driver of IL-10 during macrophage infection with H. capsulatum in the absence of HIF-1α. Accumulation of fumarate reduced expression of a HIF-1α-dependent microRNA (miRNA), miR-27a, known to mediate decay of Il10 mRNA. Inhibition of fumarate accrual in vivo limited IL-10 and fungal growth. Our data demonstrate the critical role of HIF-1α in shaping appropriate TCA cycle activity in response to infection and highlight the consequences of a dysregulated immunometabolic response. IMPORTANCE Histoplasma capsulatum and related Histoplasma species are intracellular fungal pathogens endemic to broad regions of the globe, including the Americas, Africa, and Asia. While most infections resolve with mild or no symptoms, failure of the host to control fungal growth produces severe disease. Previously, we reported that loss of a key transcriptional regulator, hypoxia-inducible factor 1α (HIF-1α), in macrophages led to a lethal failure to control growth of Histoplasma (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565–579, 2016, https://doi.org/10.4049/jimmunol.1600342). Inhibition of phagocyte activation due to excessive interleukin 10 by HIF-1α-deficient macrophages drove this outcome. In this study, we demonstrate that HIF-1α maintains contextually appropriate TCA cycle metabolism within Histoplasma-infected macrophages. The absence of HIF-1α results in excessive fumarate production that alters miRNA-27a regulation of interleukin-10. HIF-1α thus preserves the capacity of macrophages to transition from a permissive intracellular niche to the site of pathogen killing.

Macrophages are indispensable immune cells tasked at eliminating intracellular pathogens. Mycobacterium tuberculosis (Mtb), one of the most virulent intracellular bacterial pathogens known to man, infects and resides within macrophages. While macrophages can be provoked by extracellular stimuli to inhibit and kill Mtb bacilli, these host defense mechanisms can be blocked by limiting nutritional metabolites, such as amino acids. The amino acid L-arginine has been well described to enhance immune function, especially in the context of driving macrophage nitric oxide (NO) production in mice. In this study, we aimed to establish the necessity of L-arginine on anti-Mtb macrophage function independent of NO. Utilizing an in vitro system, we identified that macrophages relied on NO for only half of their L-arginine-mediated host defenses and this L-arginine-mediated defense in the absence of NO was associated with enhanced macrophage numbers and viability. Additionally, we observed macrophage glycolysis to be driven by both L-arginine and mechanistic target of rapamycin (mTOR), and inhibition of glycolysis or mTOR reduced macrophage control of Mtb as well as macrophage number and viability in the presence of L-arginine. Our data underscore L-arginine as an essential nutrient for macrophage function, not only by fueling anti-mycobacterial NO production, but also as a central regulator of macrophage metabolism and additional host defense mechanisms.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death worldwide. Despite decades of research, there is still much to be uncovered regarding the immune response to Mtb infection. Here, we summarize the current knowledge on anti-Mtb immunity, with a spotlight on immune cell amino acid metabolism. Specifically, we discuss L-arginine and L-tryptophan, focusing on their requirements, regulatory roles, and potential use as adjunctive therapy in TB patients. By continuing to uncover the immune cell contribution during Mtb infection and how amino acid utilization regulates their functions, it is anticipated that novel host-directed therapies may be developed and/or refined, helping to eradicate TB.

Tuberculosis has been associated with increased risk of atherosclerotic cardiovascular disease. To examine whether mycobacterial infection exacerbates atherosclerosis development in experimental conditions, we infected low-density lipoprotein receptor knockout (Ldlr-/-) mice with Mycobacterium bovis Bacille-Calmette-Guérin (BCG), an attenuated strain of the Mycobacterium tuberculosis complex. Twelve-week old male Ldlr-/- mice were infected with BCG (0.3–3.0x106 colony-forming units) via the intranasal route. Mice were subsequently fed a western-type diet containing 21% fat and 0.2% cholesterol for up to 16 weeks. Age-matched uninfected Ldlr-/- mice fed with an identical diet served as controls. Atherosclerotic lesions in aorta were examined using Oil Red O staining. Changes induced by BCG infection on the immunophenotyping profile of circulating T lymphocytes and monocytes were assessed using flow cytometry. BCG infection increased atherosclerotic lesions in en face aorta after 8 weeks (plaque ratio; 0.021±0.01 vs. 0.013±0.01; p = 0.011) and 16 weeks (plaque ratio, 0.15±0.13 vs. 0.06±0.02; p = 0.003). No significant differences in plasma cholesterol or triglyceride levels were observed between infected and uninfected mice. Compared to uninfected mice, BCG infection increased systemic CD4/CD8 T cell ratio and the proportion of Ly6Clow non-classical monocytes at weeks 8 and 16. Aortic plaque ratios correlated with CD4/CD8 T cell ratios (Spearman’s rho = 0.498; p = 0.001) and the proportion of Ly6Clow non-classical monocytes (Spearman’s rho = 0.629; p < 0.001) at week 16. In conclusion, BCG infection expanded the proportion of CD4+ T cell and Ly6Clow monocytes, and aggravated atherosclerosis formation in the aortas of hyperlipidemic Ldlr-/- mice. Our results indicate that mycobacterial infection is capable of enhancing atherosclerosis development.

Obesity is a prevalent predisposing factor to non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease in the developed world. NAFLD spectrum of disease involves progression from steatosis (NAFL), to steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Despite clinical and public health significance, current FDA approved therapies for NAFLD are lacking in part due to insufficient understanding of pathogenic mechanisms driving disease progression. The etiology of NAFLD is multifactorial. The induction of both systemic and tissue inflammation consequential of skewed immune cell metabolic state, polarization, tissue recruitment, and activation are central to NAFLD progression. Here, we review the current understanding of the above stated cellular and molecular processes that govern macrophage contribution to NAFLD pathogenesis and how adipose tissue and liver crosstalk modulates macrophage function. Notably, the manipulation of such events may lead to the development of new therapies for NAFLD.

Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs) infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

Activation, recruitment, and effector function of T lymphocytes are essential for control of mycobacterial infection. These processes are tightly regulated in T cells by the availability of l-arginine within the microenvironment. In turn, mycobacterial infection dampens T cell responsiveness through arginase induction in myeloid cells, promoting sequestration of l-arginine within the local milieu. Here, we show T cells can replenish intracellular l-arginine through metabolism of l-citrulline to mediate inflammatory function, allowing anti-mycobacterial T cells to overcome arginase-mediated suppression. Furthermore, T cell l-citrulline metabolism is necessary for accumulation of CD4+ T cells at the site of infection, suggesting this metabolic pathway is involved during anti-mycobacterial T cell immunity in vivo. Together, these findings establish a contribution for l-arginine synthesis by T cells during mycobacterial infection, and implicate l-citrulline as a potential immuno-nutrient to modulate host immunity.

OBJECTIVES/SPECIFIC AIMS: The study aimed at assessing whether M. bovis BCG infection and inflammation exacerbates the development of atherosclerosis in Ldlr-/- mice. METHODS/STUDY POPULATION: Twelve-week old male Ldlr-/- mice (n=10) were infected with M. bovis BCG (0.3–3.0x10^6 colony-forming units (CFUs)) via the intranasal route, to simulate a natural respiratory route of infection. Mice were subsequently fed a western-type diet (WD) containing 21% fat and 0.2% cholesterol for 16 weeks. Age-matched uninfected Ldlr-/- mice (n=10) fed with an identical WD served as controls. Mice were euthanized after 16 weeks of WD to examine atherosclerotic lesions in aortic root sections and en face aorta using Oil Red O staining. Plasma cholesterol and triglyceride levels were measured by enzymatic assays and lipoprotein distribution was assessed using fast protein liquid chromatography. Because of the important role of T cells and monocytes in atherosclerosis development, we assessed these cell subsets in blood using flow cytometry at 8 and 16 weeks. Experiments were conducted in duplicate. We used unpaired Student’s t-test for group comparisons of numeric variables and flow cytometry data. RESULTS/ANTICIPATED RESULTS: M. bovis BCG infection significantly increased atherosclerotic lesions in en face aorta (plaque size per aorta area ratio; 0.15±0.13 vs. 0.06±0.02; P

Despite the clear importance of the left-handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix-forming propensities measured for all residues, except tyrosine and tryptophan, in a proline-based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix-forming propensity scale will prove useful in future studies of the conformational properties of proline-rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states. Proteins 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.