Cutting Edge: l-Arginine Transfer from Antigen-Presenting Cells Sustains CD4+ T Cell Viability and Proliferation.

Metabolomics analyses suggest changes in amino acid abundance, particularly l-arginine (L-ARG), occur in patients with tuberculosis. Immune cells require L-ARG to fuel effector functions following infection. We have previously described an L-ARG synthesis pathway in immune cells; however, its role in APCs has yet to be uncovered. Using a coculture system with mycobacterial-specific CD4+ T cells, we show APC L-ARG synthesis supported T cell viability and proliferation, and activated T cells contained APC-derived L-ARG. We hypothesize that APCs supply L-ARG to support T cell activation under nutrient-limiting conditions. This work expands the current model of APC-T cell interactions and provides insight into the effects of nutrient availability in immune cells.

l-Arginine Synthesis from l-Citrulline in Myeloid Cells Drives Host Defense against Mycobacteria In Vivo.

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

Microbicidal NO production is reliant on inducible NO synthase-mediated L-arginine metabolism in macrophages (MΦs). However, L-arginine supply can be restricted by arginase activity, resulting in inefficient NO output and inhibition of antimicrobial MΦ function. MΦs circumvent this by converting L-citrulline to L-arginine, thereby resupplying substrate for NO production. In this article, we define the metabolic signature of mycobacteria-infected murine MΦs supplied L-arginine, L-citrulline, or both amino acids. Using liquid chromatography-tandem mass spectrometry, we determined that L-arginine synthesized from L-citrulline was less effective as a substrate for arginase-mediated L-ornithine production compared with L-arginine directly imported from the extracellular milieu. Following Mycobacterium bovis bacillus Calmette-Guérin infection and costimulation with IFN-γ, we observed that MΦ arginase activity did not inhibit production of NO derived from L-citrulline, contrary to NO inhibition witnessed when MΦs were cultured in L-arginine. Furthermore, we found that arginase-expressing MΦs preferred L-citrulline over L-arginine for the promotion of antimycobacterial activity. We expect that defining the consequences of L-citrulline metabolism in MΦs will provide novel approaches for enhancing immunity, especially in the context of mycobacterial disease.

Water depth influences survival and predator-specific patterns of nest loss in three secretive marsh bird species.

Wetlands have become increasingly rare in the United States, negatively influencing wetland-dependent birds, and many remaining wetlands are intensively managed through seasonal dewatering mimicking historic flood pulses during spring and summer. However, water around nests may provide protection from terrestrial predators, and lowering water levels during the breeding season of wetland birds may increase predation risk and exacerbate marsh bird population declines. Understanding interactions between water depth, nesting marsh birds, and nest predators is critical to aid managers in developing a multi-species management approach in emergent wetlands. During the 2020 and 2021 breeding seasons, we examined nest survival of 148 marsh bird nests (American Coot, Fulica americana, n = 1; Common Gallinule, Gallinula galeata, n = 64; and Least Bittern; Ixobrychus exilis, n = 83) and installed cameras at 78 nests to identify predators at a large, restored floodplain wetland in Illinois where the primary management technique is seasonal water removal to stimulate germination of moist soil plants. We found nest predation of, and abandonment by, Least Bittern and Common Gallinule were related to shallower water, and early season, high volume dewatering. Least Bitterns nested more commonly along wetland edges and nests farther from the shore were more likely to survive. Similarly, we found mammalian depredation of nests and nest abandonment decreased when deeper water was present around nests. Alternatively, snake predation was observed earlier in the year prior to water removal from inundated emergent vegetation. Our results demonstrate water depth may be an important deterrent of nest predators, especially mammals, during the breeding season. Further, we recommend managers delay dewatering until after the nesting season at sites where management for conservation-priority marsh birds is a focus.

Promotion of Anti-Tuberculosis Macrophage Activity by L-Arginine in the Absence of Nitric Oxide.

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.

A new Miopsalis from Mindanao supports a biogeographic umbilicus between Borneo and the southern Philippines (Arachnida: Opiliones: Cyphophthalmi: Stylocellidae).

A new species of Cyphophthalmi belonging to the Southeast Asian genus Miopsalis Thorell, 1890 is described and illustrated using SEM and confocal microscopy. The species is known from two localities in the northern part of Mindanao and constitutes only the second described Cyphophthalmi species endemic to the Philippines. Sequence data obtained from this species show that it is phylogenetically distinct from two juvenile specimens previously collected from Bangaray Kimlawis (southern Mindanao) in 2009. The new Miopsalis is clearly distinguishable from other Miopsalis by characters pertaining to spermatopositor morphology.

Mycobacterium bovis Bacille-Calmette-Guérin Infection Aggravates Atherosclerosis.

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.

Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis.

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

l-Citrulline Metabolism in Mice Augments CD4+ T Cell Proliferation and Cytokine Production In Vitro, and Accumulation in the Mycobacteria-Infected Lung.

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.

Effect of oscillating fluid shear on solute transport in cortical bone.

The consequences of an oscillatory fluid shear mechanism on nutrient transport in bone during physical activity and ultrasonic therapy are discussed. During movement, periodic stress on bone creates transient pressure gradients that circulate interstitial fluid through calcified bone. A transport model derived from oscillatory Taylor-Aris dispersion phenomena was used to predict a ratio of effective-to-molecular diffusivity, K/D, for solutes of varying sizes up to 50 nm in diameter, in pores filled with interstitial fluid and pericellular matrix. The magnitude of the estimated transport enhancement depended on the molecular size, pore dimension, applied frequency and the displacement of the fluid during pressurization. For oscillation frequencies and amplitudes corresponding to those experienced during normal human activity, transport enhancements of up to 100 fold are expected for molecules larger than 5 nm in diameter. Enhancements of up to one order of magnitude, due to ultrasound stimulations in the MHz frequency range, are also expected for 7-nm-sized solutes. No effects are anticipated for ions, whose molecular diffusion time is too fast relative to the oscillation frequency. This model is expected to be useful for understanding differences in bone growth as a function of type of movement or to develop new physical therapies.

Rapamycin Regulates Bleomycin-Induced Lung Damage in SP-C-Deficient Mice.

Injury to the distal respiratory epithelium has been implicated as an underlying cause of idiopathic lung diseases. Mutations that result in SP-C deficiencies are linked to a small subset of spontaneous and familial cases of interstitial lung disease (ILD) and interstitial pulmonary fibrosis (IPF). Gene-targeted mice that lack SP-C (Sftpc(-/-)) develop an irregular ILD-like disease with age and are a model of the human SP-C related disease. In the current study, we investigated whether rapamycin could ameliorate bleomycin-induced fibrosis in the lungs of Sftpc(-/-) mice. Sftpc(+/+) and -/- mice were exposed to bleomycin with either preventative administration of rapamycin or therapeutic administration beginning eight days after the bleomycin injury. Rapamycin-treatment increased weight loss and decreased survival of bleomycin-treated Sftpc(+/+) and Sftpc(-/-) mice. Rapamycin did not reduce the fibrotic disease in the prophylactic or rescue experiments of either genotype of mice. Further, rapamycin treatment augmented airway resistance and reduced lung compliance of bleomycin-treated Sftpc(-/-) mice. Rapamycin treatment was associated with an increased expression of profibrotic Th2 cytokines and reduced expression of INF-γ. These findings indicate that novel therapeutics will be required to treat individuals with SP-C deficient ILD/IPF.

Uptake of calcium phosphate nanoshells by osteoblasts and their effect on growth and differentiation.

The influence of calcium phosphate nanoshell materials on the uptake, viability, and mineralization of human fetal osteoblast cultures was evaluated. Proliferation rates and alkaline phosphatase activity of the cultures were unaffected by the addition of nanoshells to the growth media, but mineralization levels were enhanced by nearly 40%, in contrast to media prepared without nanoshells, or with other calcium phosphate nanomaterials. Nanoshells were internalized by macropinocytosis, and migrated toward the cell nucleus at a rate of 0.34 microm hr(-1). Dye-loaded nanoshells maintained high light emission intensity for over five days while inside the cells, where they could be used as intracellular markers for in vitro microscopic imaging. From these results, it appears that the CaP nanoshells could be developed into a safe sensor and delivery vehicle for osteoblast cell culture studies, whereas the carrier itself has intrinsic bioactivity and may itself upregulate the formation of new bone.