Dennis Montoya

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.

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.

Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

Divergence of macrophage phagocytic and antimicrobial programs in leprosy

Effective innate immunity against many microbial pathogens requires macrophage programs that upregulate phagocytosis and direct antimicrobial pathways, two functions generally assumed to be coordinately regulated. We investigated the regulation of these key functions in human blood-derived macrophages. Interleukin-10 (IL-10) induced the phagocytic pathway, including the C-type lectin CD209 and scavenger receptors, resulting in phagocytosis of mycobacteria and oxidized low-density lipoprotein. IL-15 induced the vitamin D-dependent antimicrobial pathway and CD209, yet the cells were less phagocytic. The differential regulation of macrophage functional programs was confirmed by analysis of leprosy lesions: the macrophage phagocytosis pathway was prominent in the clinically progressive, multibacillary form of the disease, whereas the vitamin D-dependent antimicrobial pathway predominated in the self-limited form and in patients undergoing reversal reactions from the multibacillary to the self-limited form. These data indicate that macrophage programs for phagocytosis and antimicrobial responses are distinct and differentially regulated in innate immunity to bacterial infections.

Identification of Prostaglandin E2 Receptor Subtype 2 As a Receptor Activated by OxPAPC

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial &bgr;1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein–coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of &bgr;1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor-&agr; and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

IL-32 is a molecular marker of a host defense network in human tuberculosis

IL-32 links the microbicidal peptide pathway with resistance to disease progression in clinical tuberculosis. TB Sunny Side Up Interleukin-32 (IL-32) could be a correlate of protection against tuberculosis. Montoya et al. report that IL-32 is a potential candidate marker of host defense against tuberculosis. Mycobacterium tuberculosis causes a sometimes deadly infection that nonetheless only develops to disease in about 10% of individuals. The authors performed gene expression profiles of human macrophages and found an association between IL-32 and the vitamin D antimicrobial pathway. Moreover, analysis of five different clinical data sets suggested that IL-32 can serve as a molecular marker of latent tuberculosis and may be activated in response to signaling by IL-15. These data suggest that IL-32 may not only serve as a putative correlate of protection but also contribute directly to host response to tuberculosis. Tuberculosis is a leading cause of infectious disease–related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ– and IL-15–induced “defense response” genes. IL-32 induced the vitamin D–dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15–induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15–induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis.

Learning from Leprosy: Insight into the Human Innate Immune Response

Investigation into the innate immune response in leprosy has provided insight into host defense and immunopathology in human infectious disease. A key advance has been the delineation of pattern recognition receptors that detect pathogen-associated molecular patterns of the bacterium that causes leprosy, Mycobacterium leprae. From this knowledge, it has been possible to determine the cytokine responses as well as macrophage and dendritic cell differentiation programs that contribute to host defense and tissue injury in leprosy. These insights provide targets for therapeutic intervention to modulate innate immune responses against microbial infection in humans.

Interleukin‐1β triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to T cells

The rapid differentiation of monocytes into macrophages (MΦ) and dendritic cells is a pivotal aspect of the innate immune response. Differentiation is triggered following recognition of microbial ligands that activate pattern recognition receptors or directly by pro‐inflammatory cytokines. We demonstrate that interleukin‐1β (IL‐1β) induces the rapid differentiation of monocytes into CD209+ MΦ, similar to activation via Toll‐like receptor 2/1, but with distinct phenotypic and functional characteristics. The IL‐1β induced MΦ express higher levels of key markers of phagocytosis, including the Fc‐receptors CD16 and CD64, as well as CD36, CD163 and CD206. In addition, IL‐1β‐induced MΦ exert potent phagocytic activity towards inert particles, oxidized low‐density lipoprotein and mycobacteria. Furthermore, IL‐1β‐induced MΦ express higher levels of HLA‐DR and effectively present mycobacterial antigens to T cells. Therefore, the ability of IL‐1β to induce monocyte differentiation into MΦ with both phagocytosis and antigen‐presenting function is a distinct part of the innate immune response in host defence against microbial infection.

Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients

We have investigated transcriptional and epigenetic differences in peripheral blood mononuclear cells (PBMCs) of monozygotic female twins discordant in the diagnosis of amyotrophic lateral sclerosis (ALS). Exploring DNA methylation differences by reduced representation bisulfite sequencing (RRBS), we determined that, over time, the ALS twin developed higher abundances of the CD14 macrophages and lower abundances of T cells compared to the non‐ALS twin. Higher macrophage signature in the ALStwinwas also shownby RNA sequencing (RNA‐seq). Moreover, the twins differed in the methylome at loci near several genes, including EGFR and TNFRSF11A, and in the pathways related to the tretinoin and H3K27me3 markers. We also tested cytokine production by PBMCs. The ALS twins PBMCs spontaneously produced IL‐6 and TNF‐α, whereas PBMCs of the healthy twin produced these cytokines only when stimulated by superoxide dismutase (SOD)‐1. These results and flow cytometric detection of CD45 and CD127 suggest the presence of memory T cells in both twins, but effector T cells only in the ALS twin. The ALS twins PBMC supernatants, but not the healthy twins, were toxic to rat cortical neurons, and this toxicity was strongly inhibited by an IL‐6 receptor antibody (tocilizumab) and less well by TNF‐α and IL‐1β antibodies. The putative neurotoxicity of IL‐6 and TNF‐α is in agreement with a high expression of these cytokines on infiltrating macrophages in the ALS spinal cord. We hypothesize that higher macrophage abundance and increased neurotoxic cytokines have a fundamental role in the phenotype and treatment of certain individuals with ALS.—Lam, L., Chin, L., Halder, R.C., Sagong, B., Famenini, S., Sayre, J., Montoya, D., Rubbi L., Pellegrini, M., Fiala, M. Epigenetic changes in T‐cell and monocyte signatures and production of neurotoxic cytokines in ALS patients. FASEB J. 30, 3461–3473 (2016). www.fasebj.org

Genetic Mechanisms of Immune Evasion in Colorectal Cancer

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730-49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.