P'ng Loke

PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells

PD-L1 and PD-L2 are ligands for PD-1, a costimulatory molecule that plays an inhibitory role in regulating T cell activation in the periphery. We find that PD-L1 is highly expressed on inflammatory macrophages as compared with resident peritoneal macrophages but can be induced on resident macrophages by classical activation stimuli such as lipopolysaccharide, IFN-γ, and polyinosinic-polycytidylic acid. Further up-regulation of PD-L1 on inflammatory macrophages can also be induced by subsequent exposure to lipopolysaccharide and IFN-γ. In contrast, PD-L2 is not expressed on inflammatory macrophages but can be induced by alternative activation via IL-4. Although PD-L1 is highly inducible on a variety of antigen-presenting cell lines as well as resident macrophages, PD-L2 is most significantly inducible only on inflammatory macrophages. PD-L1 up-regulation depends on TLR4 and STAT1, whereas PD-L2 expression depends on IL-4Rα and STAT6. Consistent with these results, T helper 1/T helper 2 (Th1/Th2) cells also differentially up-regulate PD-L1 and PD-L2 expression on inflammatory macrophages. Hence, Th1 cells as well as microbial products can enhance PD-L1 expression on many different macrophage populations, whereas Th2 cells instruct only inflammatory macrophages to up-regulate PD-L2. These results suggest that PD-L1 and PD-L2 might have different functions in regulating type 1 and type 2 responses.

Tryptophan Catabolism by Indoleamine 2,3-Dioxygenase 1 Alters the Balance of TH17 to Regulatory T Cells in HIV Disease

Patients with AIDS have fewer immune cells to defend against microbial invasion through the gut, a critical loss that may be caused by a tryptophan metabolite produced by other immune cells. Loss of the Defenders at the Gate Like archers stationed along the walls of a medieval castle, the immune system patrols the vulnerable parts of our body to keep pathogens at bay. One of these susceptible areas is the mucosa of the gastrointestinal tract, which is continually exposed to ingested and resident pathogens. This defense breaks down in patients with AIDS, in which sentinel immune cells [T helper 17 (TH17) cells] are missing from the gastrointestinal lining, potentially accounting for some secondary infections acquired by these patients. Favre and colleagues present evidence that the loss of these cells (and a parallel increase in immune suppressor cells) is caused by a metabolite of the amino acid tryptophan, new understanding that should help to prevent this serious consequence of HIV infection. HIV disease is in part an inflammatory disease, and activated T cells and cytokines circulate in patients’ blood, along with pathogen-derived molecules that trigger the innate immune system. The authors show that, in patients with serious AIDS, who are in this inflammatory state, the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which catabolizes tryptophan, is elevated in dendritic cells (DCs)—agents that present antigen to the immune system—from the blood, lymph nodes, and mucosa of the lower gastrointestinal tract. The inflammation-related molecules interferon γ and bacterial lipopolysaccharide can induce IDO1 in isolated DCs. This excess IDO1 activation increased blood concentrations of tryptophan catabolites in patients, and two of the catabolites increased the proportion of TH17 (activating) immune cells and decreased the proportion of T regulatory (Treg) (suppressing) immune cells in culture. In patients with serious disease, the authors found that the ratio of TH17 to Treg cells was much lower than normal, which hampers the ability of the body to raise an effective immune defense against pathogens. This dysfunctional system would set up a reinforcing loop that progressively depletes vulnerable tissues of their immune protection. Paradoxically, it seems, activation of the immune system by HIV may be contributing to the decline in immune function that is the hallmark of the disease. IDO1 inhibitors are being tested for their efficacy in interfering with this dangerous depletion of defenses. The pathogenesis of human and simian immunodeficiency viruses is characterized by CD4+ T cell depletion and chronic T cell activation, leading ultimately to AIDS. CD4+ T helper (TH) cells provide protective immunity and immune regulation through different immune cell functional subsets, including TH1, TH2, T regulatory (Treg), and interleukin-17 (IL-17)–secreting TH17 cells. Because IL-17 can enhance host defenses against microbial agents, thus maintaining the integrity of the mucosal barrier, loss of TH17 cells may foster microbial translocation and sustained inflammation. Here, we study HIV-seropositive subjects and find that progressive disease is associated with the loss of TH17 cells and a reciprocal increase in the fraction of the immunosuppressive Treg cells both in peripheral blood and in rectosigmoid biopsies. The loss of TH17/Treg balance is associated with induction of indoleamine 2,3-dioxygenase 1 (IDO1) by myeloid antigen-presenting dendritic cells and with increased plasma concentration of microbial products. In vitro, the loss of TH17/Treg balance is mediated directly by the proximal tryptophan catabolite from IDO metabolism, 3-hydroxyanthranilic acid. We postulate that induction of IDO may represent a critical initiating event that results in inversion of the TH17/Treg balance and in the consequent maintenance of a chronic inflammatory state in progressive HIV disease.

B7x: A widely expressed B7 family member that inhibits T cell activation

B7 family proteins provide costimulatory signals that regulate T cell responses. Here we report the third set of B7 family-related T cell inhibitory molecules with the identification of a homolog of the B7 family, B7x. It is expressed in immune cells, nonlymphoid tissues, and some tumor cell lines. B7x inhibits cell-cycle progression, proliferation, and cytokine production of both CD4+ and CD8+ T cells. B7x binds a receptor that is expressed on activated, but not resting T cells that is distinct from known CD28 family members. Its receptor may be a recently identified inhibitory molecule, B and T lymphocyte attenuator. These studies identify a costimulatory pathway that may have a unique function in downregulation of tissue-specific autoimmunity and antitumor responses.

Alternative activation is an innate response to injury that requires CD4+ T cells to be sustained during chronic infection.

Alternatively activated macrophages (AAMΦ) are found in abundance during chronic Th2 inflammatory responses to metazoan parasites. Important roles for these macrophages are being defined, particularly in the context of Th2-mediated pathology and fibrosis. However, a full understanding of the requirements for alternative activation, particularly at the innate level, is lacking. We present evidence that alternative activation by the Th2 cytokines IL-4 and IL-13 is an innate and rapid response to tissue injury that takes place even in the absence of an infectious agent. This early response does not require CD4+ Th2 cells because it occurred in RAG-deficient mice. However, class II-restricted CD4+ T cell help is essential to maintain AAMΦ in response to infection, because AAMΦ were absent in RAG-deficient and MHC class II-deficient, but not B cell-deficient mice after chronic exposure to the nematode parasite, Brugia malayi. The absence of AAMΦ was associated with increased neutrophilia and reduced eosinophilia, suggesting that AAMΦ are involved in the clearance of neutrophils as well as the recruitment of eosinophils. Consistent with this hypothesis, AAMΦ show enhanced phagocytosis of apoptotic neutrophils, but not latex beads. Our data demonstrate that alternative activation by type 2 cytokines is an innate response to injury that can occur in the absence of an adaptive response. However, analogous to classical activation by microbial pathogens, Th2 cells are required for maintenance and full activation during the ongoing response to metazoan parasites.

Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells

ABSTRACT Ym1 and Fizz1 are secreted proteins that have been identified in a variety of Th2-mediated inflammatory settings. We originally found Ym1 and Fizz1 as highly expressed macrophage genes in a Brugia malayi infection model. Here, we show that their expression is a generalized feature of nematode infection and that they are induced at the site of infection with both the tissue nematode Litomosoides sigmodontis and the gastrointestinal nematode Nippostrongylus brasiliensis. At the sites of infection with N. brasiliensis, we also observed induction of other chitinase and Fizz family members (ChaFFs): acidic mammalian chitinase (AMCase) and Fizz2. The high expression of both Ym1 and AMCase in the lungs of infected mice suggests that abundant chitinase production is an important feature of Th2 immune responses in the lung. In addition to expression of ChaFFs in the tissues, Ym1 and Fizz1 expression was observed in the lymph nodes. Expression both in vitro and in vivo was restricted to antigen-presenting cells, with the highest expression in B cells and macrophages. ChaFFs may therefore be important effector or wound-repair molecules at the site of nematode infection, with potential regulatory roles for Ym1 and Fizz1 in the draining lymph nodes.

Alternatively activated macrophages induced by nematode infection inhibit proliferation via cell‐to‐cell contact

The cytokine microenvironment is thought to play an important role in the generation of immunoregulatory cells. Nematode infections are commonly associated with Th2 cytokines and hyporesponsive T cells. Here we show that IL‐4‐dependent macrophages recruited in vivo by the nematode parasite Brugia malayi actively suppress the proliferation of lymphocytes on co‐culture in vitro. These alternatively activated macrophages block proliferation by cell‐to‐cell contact, implicating a receptor‐mediated mechanism. Further, the proliferative block is reversible and is not a result of apoptosis. Suppressed cells accumulate in the G1 and G2 / M phase of the cell cycle. Interestingly, the G1 and G2 / M block correlates with increased levels of Ki‐67 protein, suggesting a mechanism that affects degradation of cell cycle proteins. We also show that, in addition to lymphocyte cell lines of murine origin, these suppressive cells can inhibit proliferation of a wide range of transformed human carcinoma lines. Our data reveal a novel mechanism of proliferative suppression induced by a parasitic nematode that acts via IL‐4‐dependent macrophages. These macrophages may function as important immune regulatory cells in both infectious and noninfectious disease contexts.

Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota

Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota.

A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection

Infections with the helminth parasite Brugia malayi share many key features with Th2-mediated allergic diseases, including recruitment of eosinophils. We have investigated the dynamics of inflammatory cell recruitment under type 2 cytokine conditions in mice infected with B. malayi. Among the cells recruited to the site of infection is a novel population of “alternatively activated” macrophages that ablate cell proliferation and enhance Th2 differentiation. By profiling gene expression in this macrophage population, we found a dramatic up-regulation of a recently described eosinophil chemotactic factor, eosinophil chemotactic factor-L/Ym1, representing over 9% of clones randomly selected from a cDNA library. Because B. malayi is known to secrete homologs (Bm macrophage migration inhibitory factor (MIF)-1 and -2) of the human cytokine MIF, we chose to investigate the role this cytokine mimic may play in the development of the novel macrophage phenotype observed during infection. Strikingly, administration of soluble recombinant Bm-MIF-1 was able to reproduce the effects of live parasites, leading both to the up-regulation of Ym1 by macrophages and a marked recruitment of eosinophils in vivo. Because activity of Bm-MIF-1 is dependent upon an amino-terminal proline, this residue was mutated to glycine; the resultant recombinant (Bm-MIF-1G) was unable to induce Ym1 transcription in macrophages or to mediate the recruitment of eosinophils. These data suggest that macrophages may provide a crucial link between helminth parasites, their active cytokine mimics, and the recruitment of eosinophils in infection.

Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct

Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. AAMs can be derived either from proliferation of tissue resident macrophages or recruited inflammatory monocytes, but it is not known whether these different sources generate AAMs that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that, although both monocyte and tissue-derived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green fluorescent protein (GFP)(high) and expressed CD206, whereas tissue-derived AAMs were CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde dehydrogenase activity and promoted the differentiation of FoxP3(+) cells from naïve CD4(+) cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with immune regulation, and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells.

Therapeutic Helminth Infection of Macaques with Idiopathic Chronic Diarrhea Alters the Inflammatory Signature and Mucosal Microbiota of the Colon

Idiopathic chronic diarrhea (ICD) is a leading cause of morbidity amongst rhesus monkeys kept in captivity. Here, we show that exposure of affected animals to the whipworm Trichuris trichiura led to clinical improvement in fecal consistency, accompanied by weight gain, in four out of the five treated monkeys. By flow cytometry analysis of pinch biopsies collected during colonoscopies before and after treatment, we found an induction of a mucosal T(H)2 response following helminth treatment that was associated with a decrease in activated CD4(+) Ki67+ cells. In parallel, expression profiling with oligonucleotide microarrays and real-time PCR analysis revealed reductions in T(H)1-type inflammatory gene expression and increased expression of genes associated with IgE signaling, mast cell activation, eosinophil recruitment, alternative activation of macrophages, and worm expulsion. By quantifying bacterial 16S rRNA in pinch biopsies using real-time PCR analysis, we found reduced bacterial attachment to the intestinal mucosa post-treatment. Finally, deep sequencing of bacterial 16S rRNA revealed changes to the composition of microbial communities attached to the intestinal mucosa following helminth treatment. Thus, the genus Streptophyta of the phylum Cyanobacteria was vastly increased in abundance in three out of five ICD monkeys relative to healthy controls, but was reduced to control levels post-treatment; by contrast, the phylum Tenericutes was expanded post-treatment. These findings suggest that helminth treatment in primates can ameliorate colitis by restoring mucosal barrier functions and reducing overall bacterial attachment, and also by altering the communities of attached bacteria. These results also define ICD in monkeys as a tractable preclinical model for ulcerative colitis in which these effects can be further investigated.