Shankar S. Iyer

The Nalp3 inflammasome is essential for the development of silicosis

Inhalation of crystalline silica and asbestos is known to cause the progressive pulmonary fibrotic disorders silicosis and asbestosis, respectively. Although alveolar macrophages are believed to initiate these inflammatory responses, the mechanism by which this occurs has been unclear. Here we show that the inflammatory response and subsequent development of pulmonary fibrosis after inhalation of silica is dependent on the Nalp3 inflammasome. Stimulation of macrophages with silica results in the activation of caspase-1 in a Nalp3-dependent manner. Macrophages deficient in components of the Nalp3 inflammasome were incapable of secreting the proinflammatory cytokines interleukin (IL)-1β and IL-18 in response to silica. Similarly, asbestos was capable of activating caspase-1 in a Nalp3-dependent manner. Activation of the Nalp3 inflammasome by silica required both an efflux of intracellular potassium and the generation of reactive oxygen species. This study demonstrates a key role for the Nalp3 inflammasome in the pathogenesis of pneumoconiosis.

Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.

Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1β. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.

Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.

Vitamin D is required for both innate and adaptive immunity to tuberculosis. The Sunny Side of Antimicrobial Response Nearly one-third of the world’s population is thought to be infected with Mycobacterium tuberculosis, which causes a potentially fatal lung disease in untreated patients. Although most M. tuberculosis infections can be treated by antibiotic therapy, the burden of infection is especially high in immunodeficient (HIV+) patients and individuals from developing nations. Moreover, drug-resistant M. tuberculosis is increasingly prevalent. Yet, most humans with M. tuberculosis infection are asymptomatic, perhaps because of successful immunological control. Understanding the mechanisms behind immune control of M. tuberculosis infection may pinpoint potential new therapeutic avenues. Now, Fabri et al. examine the antimicrobial function of M. tuberculosis–infected human macrophages. The authors found that cells from the adaptive immune system—T cells—governed bacterial control by releasing the cytokine interferon-γ (IFN-γ), which then activated infected macrophages, inciting the cells to attack the invading M. tuberculosis. This activation depended on the presence of vitamin D, a fat-soluble prohormone thought to be beneficial for everything from bone health to cancer therapy. Indeed, this antimicrobial response was not seen with macrophages maintained in human sera from subjects with insufficient vitamin D levels. Vitamin D3 has been used historically to treat M. tuberculosis infection, but its effects have not been thoroughly tested in clinical trials. This study suggests that increasing serum levels of vitamin D, whether through supplementation or increased sun exposure, should improve the human immune response to M. tuberculosis and supports further testing of vitamin D in the clinic. Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

How colonization by microbiota in early life shapes the immune system

Microbial colonization of mucosal tissues during infancy plays an instrumental role in the development and education of the host mammalian immune system. These early-life events can have long-standing consequences: facilitating tolerance to environmental exposures or contributing to the development of disease in later life, including inflammatory bowel disease, allergy, and asthma. Recent studies have begun to define a critical period during early development in which disruption of optimal host-commensal interactions can lead to persistent and in some cases irreversible defects in the development and training of specific immune subsets. Here, we discuss the role of early-life education of the immune system during this “window of opportunity,” when microbial colonization has a potentially critical impact on human health and disease.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Type I Interferon Suppresses Type II Interferon–Triggered Human Anti-Mycobacterial Responses

Interfering with Interferons Infections with Mycobacteria, including Mycobacterium leprae or M. tuberculosis, vary substantially in their clinical presentation. For instance, in some cases of M. leprae, the infection is self-healing with very few lesions. In contrast, some people experience the disseminated form, where skin lesions abound and bacteria are abundant. In patients infected with M. leprae, Teles et al. (p. 1448, published online 28 February) found that the disseminated disease associates with a type I interferon gene signature, whereas the self-healing form associates with a type II interferon gene signature. In cultured cells, type I interferon and its downstream signaling cascade inhibited the antimicrobial response induced by type II interferons, providing a potential explanation for why robust disease rather than protection is seen in some cases of infection. Disseminated Mycobacterium leprae infection is associated with blockade of the antimicrobial response by type I interferons. Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D–dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ–induced macrophage vitamin D–dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.

Cutting Edge: Mycobacterium tuberculosis Blocks Ca2+ Signaling and Phagosome Maturation in Human Macrophages Via Specific Inhibition of Sphingosine Kinase

One-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca2+ that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca2+-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.

Mycobacterium tuberculosis Phagosomes Exhibit Altered Calmodulin-Dependent Signal Transduction: Contribution to Inhibition of Phagosome-Lysosome Fusion and Intracellular Survival in Human Macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca2+ ([Ca2+]c), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca2+]c that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca2+-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca2+]c resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca2+ ionophore-induced phagosomal maturation and enhanced the bacilli’s intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca2+-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.

Lipopolysaccharide-Mediated IL-10 Transcriptional Regulation Requires Sequential Induction of Type I IFNs and IL-27 in Macrophages

IL-10 is a potent anti-inflammatory molecule that regulates excessive production of inflammatory cytokines during an infection or tissue damage. Dysregulation of IL-10 is associated with a number of autoimmune diseases, and so, understanding the mechanisms by which IL-10 gene expression is regulated remains an important area of study. Macrophages represent a major source of IL-10, which is generated in response to TLR signaling as a feedback mechanism to curtail inflammatory response. In this study, we identify a signaling pathway in murine bone marrow-derived macrophages in which activation of TLR4 by LPS induces the expression of IL-10 through the sequential induction of type I IFNs followed by induction and signaling through IL-27. We demonstrate that IL-27 signaling is required for robust IL-10 induction by LPS and type I IFNs. IL-27 leads directly to transcription of IL-10 through the activation of two required transcription factors, STAT1 and STAT3, which are recruited to the IL-10 promoter. Finally, through systematic functional promoter-reporter analysis, we identify three cis elements within the proximal IL-10 promoter that play an important role in regulating transcription of IL-10 in response to IL-27.

Sphingosine Kinase 1 (SK1) Is Recruited to Nascent Phagosomes in Human Macrophages: Inhibition of SK1 Translocation by Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.