Mitochondrial C5aR1 activity in macrophages controls IL-1ß production underlying sterile inflammation.

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the "complosome," functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1ß production, both at the transcriptional level and processing of pro­IL-1ß. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1ß produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Integrins meet complement: The evolutionary tip of an iceberg orchestrating metabolism and immunity.

Immunologists have recently realized that there is more to the classic innate immune sensor systems than just mere protection against invading pathogens. It is becoming increasingly clear that such sensors, including the inflammasomes, toll-like receptors, and the complement system, are heavily involved in the regulation of basic cell physiological processes and particularly those of metabolic nature. In fact, their "non-canonical" activities make sense as no system directing immune cell activity can perform such task without the need for energy. Further, many of these ancient immune sensors appeared early and concurrently during evolution, particularly during the developmental leap from the single-cell organisms to multicellularity, and therefore crosstalk heavily with each other. Here, we will review the current knowledge about the emerging cooperation between the major inter-cell communicators, integrins, and the cell-autonomous intracellularly and autocrine-active complement, the complosome, during the regulation of single-cell metabolism. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Complement's favourite organelle-Mitochondria?

The complement system, well known for its central role in innate immunity, is currently emerging as an unexpected, cell-autonomous, orchestrator of normal cell physiology. Specifically, an intracellularly active complement system-the complosome-controls key pathways of normal cell metabolism during immune cell homeostasis and effector function. So far, we know little about the exact structure and localization of intracellular complement components within and among cells. A common scheme, however, is that they operate in crosstalk with other intracellular immune sensors, such as inflammasomes, and that they impact on the activity of key subcellular compartments. Among cell compartments, mitochondria appear to have built a particularly early and strong relationship with the complosome and extracellularly active complement-not surprising in view of the strong impact of the complosome on metabolism. In this review, we will hence summarize the current knowledge about the close complosome-mitochondria relationship and also discuss key questions surrounding this novel research area. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Complement receptor CD46 co-stimulates optimal human CD8+ T cell effector function via fatty acid metabolism.

The induction of human CD4+ Th1 cells requires autocrine stimulation of the complement receptor CD46 in direct crosstalk with a CD4+ T cell-intrinsic NLRP3 inflammasome. However, it is unclear whether human cytotoxic CD8+ T cell (CTL) responses also rely on an intrinsic complement-inflammasome axis. Here we show, using CTLs from patients with CD46 deficiency or with constitutively-active NLRP3, that CD46 delivers co-stimulatory signals for optimal CTL activity by augmenting nutrient-influx and fatty acid synthesis. Surprisingly, although CTLs express NLRP3, a canonical NLRP3 inflammasome is not required for normal human CTL activity, as CTLs from patients with hyperactive NLRP3 activity function normally. These findings establish autocrine complement and CD46 activity as integral components of normal human CTL biology, and, since CD46 is only present in humans, emphasize the divergent roles of innate immune sensors between mice and men.

Pulmonary tuberculosis patients with a vitamin D deficiency demonstrate low local expression of the antimicrobial peptide LL-37 but enhanced FoxP3+ regulatory T cells and IgG-secreting cells.

Control of human tuberculosis (TB) requires induction and maintenance of both macrophage and T cell effector functions. We demonstrate that pulmonary TB patients with a vitamin D deficiency had significantly reduced local levels of the vitamin D-inducible antimicrobial peptide LL-37 in granulomatous lesions compared to distal parenchyma from the infected lung. Instead, TB lesions were abundant in CD3(+) T cells and FoxP3(+) regulatory T cells as well as IgG-secreting CD20(+) B cells, particularly in sputum-smear positive patients with cavitary TB. Mycobacteria-specific serum IgG titers were also elevated in patients with active TB. An up-regulation of the B cell stimulatory cytokine IL-21 correlated with mRNA expression of CD20, total IgG and also IL-10 in the TB lesions. Altogether, vitamin D-deficient TB patients expressed a weak antimicrobial response but an IL-21 associated expansion of IgG-secreting B cells combined with a rise in FoxP3(+) regulatory T cells at the local site of infection.

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates.

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I-restricted CD8⁺ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8⁺ effector T-cell responses at the local site of infection in Mtb-challenged animals.

Interleukin-12p40 overexpression promotes interleukin-12p70 and interleukin-23 formation but does not affect bacille Calmette-Guérin and Mycobacterium tuberculosis clearance.

Interleukin (IL)-12p40, a subunit of IL-12p70 and IL-23, has previously been shown to inhibit IL-12p70 activity and interferon-gamma (IFN-gamma) production. However, recent evidence has suggested that the role of IL-12p40 is more complex. To study the contribution of IL-12p40 to immune responses against mycobacterial infections, we have used transgenic (tg) mice overexpressing IL-12p40 under the control of a major histocompatibility complex-II promoter. The IL-12p40 transgene was expressed during steady state at concentrations of 129 +/- 25 ng/ml of serum and 75 +/- 13 ng per spleen, while endogenous IL-12p40 was hardly detectable in control littermates. Bacille Calmette-Guérin (BCG) infection strongly induced the expression of IL-12p40 transgene in infected organs, and IL-12p40 monomeric and dimeric forms were identified in spleen of IL-12p40 tg mice. Excessive production of IL-12p40 resulted in a 14-fold increase in IL-12p70 serum levels in tg mice versus non-transgenic mice. IL-23 was also strongly elevated in the serum and spleens of IL-12p40 tg mice through BCG infection. While IFN-gamma and tumour necrosis factor protein levels were similar in IL-12p40 tg and non-transgenic mice, Th2 type immune responses were reduced in IL-12p40 tg mice. The number of BCG granulomas and macrophage expressing inducible nitric oxide synthase were similar in IL-12p40 tg and non-transgenic mice. IL-12p40 tg mice were as resistant as non-transgenic mice to BCG and Mycobacterium tuberculosis infections as they could efficiently control bacillary growth. These data show that high amounts of IL-12p40 promotes IL-12p70 and IL-23 formation, but that does not affect T helper 1 type immune responses and granuloma function, thus leading to normal mycobacterial clearance in infected organs.

Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions.

In the present study, we investigated the tuberculosis (TB) diagnostic performance of an assay on the basis of detection of TB-specific antibodies from peripheral blood mononuclear cells (PBMCs), to determine whether antibodies in lymphocyte secretions obtained from PBMCs would better reflect active disease than antibodies in serum. PBMCs from patients with and without TB cultured in various concentrations for different times were assessed. Immunoglobulin G (IgG) specific for antigen (bacille Calmette-Guérin [BCG] vaccine and purified protein derivative [PPD]) was measured in lymphocyte secretions. Patients with active TB had higher BCG- or PPD-specific IgG antibody responses than patients without TB or healthy subjects (P=.001). This method can be used as a quick diagnostic aid to facilitate rapid detection of TB cases.