Epigenetics of Trained Innate Immunity

The Scientist » News & Opinion » Daily News             

Documenting the epigenetic landscape of human innate immune cells reveals pathways essential for training macrophages.

By Ruth Williams | September 25, 2014

 

WIKIMEDIA, CHRISTOPH BOCK (MAX PLANCK INSTITUTE FOR

INFORMATICS)

Genome-wide epigenetic and transcription analyses of monocytes and macrophages have uncovered two crucial pathways driving macrophage training—a recently discovered form of innate immune memory—according to two studies published in Science today (September 25). Together with a third paper documenting the transcriptional diversity of early immune cell progenitors, the studies present the latest results from the ongoing European BLUEPRINT initiative, which aims to decipher the epigenomes of blood cells during health and disease.

 

“They did a very thorough transcriptomic and epigenomic analysis of these cells and . . . they uncover not just immunologic pathways, which would be expected, but also, interestingly, some metabolic pathways that may be important to the different immunologic phenotypes of these cells,” said Ofer Levy of Boston Children’s Hospital and Harvard Medical School who was not involved in the studies.

 

Monocytes are part of the innate immune system. They circulate in the blood, but also exit to surrounding tissues, differentiate into macrophages, and patrol the body disposing of pathogens and dead cells. Under certain conditions, macrophages can become either tolerant of pathogens or trained to react against additional infections. This training of macrophages is a recently discovered process, and aside from providing a physiological answer for some previously unexplained effects of vaccination, it also challenges the established dogma of innate immunity.

 

In humans, the immune system has two arms: innate and adaptive. The traditional view is that innate immunity is broad-acting and non-specific, while adaptive immunity establishes memories for very specific pathogens, explained Christine Stabell Benn, a professor of global health at the Statens Serum Institute in Copenhagen who also did not participate in the studies. “So, if you give a vaccine against measles you induce protective immunity against measles and nothing else.” But, she added, “what we have seen in our epidemiological studies is that vaccines [also] have non-specific effects.” The Bacille Calmette-Guérin (BCG) vaccine, for example, confers protection against a variety of infections with other microorganisms—and trained macrophages appear to be responsible. The new papers, said Stabell Benn, “are now providing the molecular mechanisms behind these epidemiological observations.”

 

Mihai Netea, a professor of medicine at Radboud University in the Netherlands and an author on two of the papers, has characterized trained macrophages in the dish, in animals and in healthy people, comparing the trained phenotype to tolerant macrophages, naive macrophages (neither trained not tolerant), and monocytes. But to get the bigger picture of what defines these different yet related cells, “I went to the group of Hank [Hendrik] Stunnenberg and asked for his help with the epigenetics,” Netea said. Stunnenberg is a professor of molecular biology at Radboud University, an author on all three papers and the coordinator of the BLUEPRINT consortium.

 

Netea, Stunnenberg, and their colleagues collected monocytes from healthy people and from them derived the three macrophage types—tolerant, trained, and naive. In these four cell types, they then analyzed genome-wide distributions of four epigenetic indicators of gene activity: DNAse hypersensitivity and three different histone modifications—trimethylation of histone H3 at lysine 4, monomethylation of histone H3 at lysine 4, and acetylation of histone H3 at lysine 27. They also analyzed genome-wide transcription and transcription factor binding.

 

Together the analyses pointed to specific genes and pathways that defined the four cell types, as well as the genes’ surrounding regulatory regions. Of particular interest was the discovery that genes associated with signaling via cyclic adenosine monophasphate (cAMP)—a molecule regulating cell metabolism, among other processes—and glycolysis—a pathway that produces energy from glucose—were specifically activated in the trained macrophages.

 

The team went on to show that these two pathways were necessary for developing the trained phenotype. Cultured monocytes in which cAMP signaling or the glycolysis pathway were inhibited exhibited impaired production of training-induced cytokines. Inhibition of cAMP or glycolysis in mice increased susceptibility to secondary infections following trained innate immunity.

 

Both training and tolerance induction in macrophages have a number of clinical implications, explained Netea. For example, too much tolerance can cause immunoparalysis—a life-threatening complication of sepsis, he said. Such patients could be helped, added Stunnenberg, “if we could turn around a paralyzed cell and activate it.” But training “can probably also in some situations be detrimental to the host,” said Stabell Benn, by potentially causing excessive inflammation, for example. Having the epigenomic information about these cells, she added, is therefore important “in the first place, to understand what is going on, and in the second place, because it offers the potential of both down-regulating over-energetic cells but also revitalizing those that have been paralyzed.”

 

L. Chen et al., “Transcriptional diversity during lineage commitment of human blood progenitors,” Science, doi: 10.1126/science.1251033, 2014.

 

S. Cheng et al., “mTOR- and HIF-1a–mediated aerobic glycolysis as metabolic basis for trained immunity,” Science, doi: 10.1126/science.1250684, 2014.

 

S. Saeed et al., “Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity,” Science, doi: 10.1126/science.1251086, 2014.

Tags

vaccination, macrophage, innate immunity, immunology, genetics & genomics, epigenetics and disease/medicine

 

Read More

Targeted Brain Cancer Vaccine

The Scientist » News & Opinion » Daily News            

Mouse study demonstrates the ability of a cancer vaccine targeted against a specific oncogenic mutation to elicit a protective anti-tumor immune response.

By Anna Azvolinsky | June 25, 2014

 

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES

A vaccine targeting a mutation found in a subset of tumors, including slow-growing brain malignancies called gliomas, can induce an immune response and prevent tumor progression in mice, according to a study published today (June 25) in Nature. Michael Platten, a neuro-oncologist at the German Cancer Research Center in Heidelberg, Germany, and his colleagues have shown in a mouse model of glioma that this peptide vaccine induces a mutation-specific immune response and can fight pre-existing tumors.

 

“This is a proof-of-principle study,” said Darell Bigner, a cancer researcher and brain tumor expert at Duke University in North Carolina. “The tumor-specific peptides [used in this study] have potential as a tumor vaccine, and should be evaluated in human clinical trials.”

 

The vaccine contains a short peptide sequence of the point mutation in the isocitrate dehydrogenase type 1 (IDH1), which is found in more than 70 percent of gliomas. “We wanted to target a tumor-specific antigen, so a frequently found mutation was an obvious choice,” said Platten.

 

The goal of cancer vaccines is to boost the immune system’s ability to recognize tumors as foreign. But so far, tumor-specific vaccines, which have mostly been tested in advanced cancer patients, have generally not been found to improve survival. Vaccinating against a single tumor-associated mutation has presented researchers with a challenge, as the spectrum of mutations change as a tumor progresses.

 

Still, targeting a so-called driver mutation—one that occurs early in tumor development and is likely to be required to sustain tumor growth—may be a viable approach. The IDH1 mutation is thought to be such a driver: it’s one of the earliest mutations to arise in gliomas, and is found in the vast majority of cells within the same tumor. “In the case of gliomas, this tumor-specific antigen may be sufficient as there is no heterogeneity [within individual tumors], to our knowledge which also made this an attractive immunotherapy target,” said Platten.

 

The researchers vaccinated mice that had a humanized version of the major histocompatibility complex (MHC), a set of cell surface molecules that are necessary to mediate specific immunity against antigens, either pathogens or tumor molecules. Because the MHC system differs between mice and humans, this humanized mouse model is a better first preclinical attempt to evaluate the potential utility of an immunotherapy prior to a first-in-human study. The immune response to the vaccine was restricted to a specific type of T cell response, the CD4-positive T cell and was able to control the IDH1 mutation expressing tumors in the mice.

 

The mice used to test the vaccine developed sarcomas rather than gliomas. “Many questions on whether this vaccine will work the same way on gliomas and other tumors with IDH1 mutations remain,” said Bigner.

 

The researchers also showed that four out of 25 patients with gliomas had innate T cell responses to the IDH1 mutations of their tumors.

 

Based on these results, Platten and colleagues in Germany are now going ahead with a small clinical trial to test the safety and immunogenicity of the vaccine in newly diagnosed IDH1-mutated glioma patients. Patients will receive chemotherapy along with the vaccine. “The tumors we are targeting are rather slow-growing . . . as opposed to very aggressive tumors,” said Platten. “This is an advantage because we have a larger time window of opportunity to induce immunity and the patients are not yet immune-compromised from prior chemotherapies.”

 

“The ultimate goal would be to target gliomas with a combination of active vaccination and a tumor microenvironment-targeted therapy,” said Platten. The vaccine could, in theory, also be effective for other tumor types that harbor the IDH1 mutation.

 

“Cancer vaccination is making a major rebound. There are many exciting trials coming up based on preclinical data,” said Drew Pardoll, an immune-oncology expert at the Johns Hopkins University School of Medicine. “Combining cancer vaccines with the new [immune checkpoint-inhibiting] antibodies is one of several exciting approaches in cancer vaccination right now.”

 

T. Schumacher et al., “A vaccine targeting mutant IDH1 induces anti-tumour immunity,” Nature, doi:10.1038/nature13387, 2014.

Tags

tumor targeting, neuroimmunology, mutation, immunotherapy, glioma, cancer vaccine, cancer and brain cancer

 

 

Read More

Blood-based tests for colorectal cancer screening

Worldwide, screening has been shown to reduce mortality and incidence of colorectal cancer. Despite its documented success, people still fail to participate and screening rates remain low in most countries. Given that patient-reported barriers include resistance to recommended fecal-based methods or endoscopy, blood-based tests have the potential to increase participation in colorectal cancer screening programmes.

by Dr Theo deVos

 

Different Stages of Colorectal Cancer

 

 

Background
Globally, colorectal cancer (CRC) is the third most common cancer in men and the second in women, with an estimated 1.36 million cases and causing an estimated 694,000 deaths in 2012 [1]. These rates are unnecessarily high since CRC is an excellent candidate for screening as evidenced by large randomized trials demonstrating reductions in mortality and incidence [reviewed in 2, 3]. Biologically, CRC usually develops slowly, going through a progression from non-cancerous polyp to cancer over a period of a decade or more. This biology readily lends itself to screening and early detection which has a significant positive impact on the effectiveness of intervention. For example, in the United States, 5-year survival is ~90 % if the tumour is confined locally when detected, ~70% if it has spread regionally, but only ~10% if distant metastases are present [4].

Colonoscopy is the predominantly recommended method for routine screening in some countries including the United States, as it enables detection and intervention in the same procedure. It is also the diagnostic follow-up for positive results of other screening tests. However, challenges with capacity and quality, financial concerns, and patient resistance have led to its lack of use as the primary screening modality in most settings. In some countries, flexible sigmoidoscopy is showing a resurgence, with reports demonstrating mortality and incidence benefits [2]. Table 1 displays a list of common CRC screening methods along with new methods coming on-line, today.

The first non-invasive tests for CRC were based on the detection of fecal occult blood (FOBT), and these have been further developed into immunological tests (FIT) using specific antibodies to detect hemoglobin. These tests are typically designed to allow patients to collect stool samples at home and ship the sample by mail to a central laboratory for testing. A newer alternative to fecal blood testing is the analysis of genetic/epigenetic markers in fecal material. This is the basis for the Cologuard test (Exact Sciences, WI, USA), a fecal DNA test recently approved by the US FDA [5]. Blood-based screening tests that measure tumour biomarkers in plasma or serum have been developed as a minimally-invasive alternative to fecal testing. DNA methylation tests based on SEPT9 have become available in Europe and are undergoing regulatory review in China. In addition, methylated SEPT9 testing is available as laboratory-developed tests (LDTs) in the USA, and a kitted version (Epi proColon®; Epigenomics AG, Germany) is currently undergoing US FDA premarket (PMA) review [6]. Another blood-based test, the ColonSentry risk test based on an expression panel is available as an LDT in the USA and in Japan.

Given the clear benefit of screening and the long standing availability of tests, the lack of participation is disappointing, and improving screening rates is a broadly accepted goal. As an example, the ‘80 by 2018’ campaign in the USA has set a goal of 80% adherence to screening guidelines by 2018 [7]. In order to meet this goal, barriers that prevent screening must be understood and overcome. There are numerous reports focused on understanding patient barriers to CRC screening. Although this is a complex issue involving costs, time, physician recommendation and several other factors, one consistent message from these studies is that the test methods themselves present barriers. Many patients are uncomfortable with all or part of the colonoscopy process and many are also uncomfortable with collecting and shipping fecal samples [8]. As a consequence, CRCs are diagnosed symptomatically in more instances than necessary, when the disease has spread beyond the primary site, resulting in greatly reduced survival rates. The availability of a screening test using a simple and common blood draw, which can be included as part of a regular check-up, has the potential to overcome some barriers and improve screening rates.

Blood-based screening
There are a number of approaches to the measurement of cancer biomarkers in the blood. The detection and quantification of circulating tumour cells represents an early approach, which was developed into a commercial system (e.g. CellSearch; Janssen Diagnostics, NJ, USA) though this analysis has not generally been used for cancer screening. Another alternative derives from the isolation and fractionation of circulating immune cells and the quantification of gene expression panels correlated with the disease by reverse-transcriptase PCR. This ‘sentinel concept’ is the basis for the ColonSentry test (GeneNews, Canada) in Table 1. A third alternative is the measurement of metabolic products by mass-spectrometry that are correlated with the presence of cancer. As an example, a commercial test (Cologic; Phenomenome, Canada) was developed based on the measurement of serum levels of GTA-446, an anti-inflammatory fatty acid. The most developed and perhaps simplest approach in this field is the measurement of cell-free genetic or epigenetic markers in plasma or serum that are highly correlated with the presence of cancer. As shown in Table 1, the methylated Septin9 biomarker and the Epi proColon® test were developed based on this approach.

Screening biomarkers in plasma and serum
The recognition that tumour DNA contains genetic and epigenetic changes that can serve as biomarkers dates back a number of decades. As reviewed recently, the list of biomarker reports for colorectal cancer grows ever longer [9]. Although numerous studies report on marker performance, the majority of studies include only a limited number of cases and controls, and only a small subset of markers have been rigorously tested in the clinical setting. Furthermore, a review of marker studies in ClinicalTrials.gov indicated very few ongoing CRC marker screening trials. Well validated markers include methylated SEPT9 described above, and the methylation of BCAT1 and IKZF1 sequences in plasma which have shown to be correlated with CRC [10] and are currently being tested in a clinical trial in Australia. There are many interesting genetic and epigenetic markers, but most await additional validation data that will support clinical utility.

Laboratory considerations for a plasma-based screening test
The basic concept outlined in Figure 1 illustrates key points associated with development of a genetic/epigenetic screening test. CRC screening from blood samples imposes rigorous demands that impact the reduction to practice for a test including: (a) high volume (millions of tests); (b) low target copy number (~1 copy per mL); (c) fragmented DNA; (d) large sample size (e.g. 3.5 mL); and (e) kitted reagents. These are discussed using the methylated Septin9 test as a case study.

Blood draw and processing
Given that screening is a high volume activity, an inexpensive and standard sample collection method is beneficial. In this case, a simple blood draw using a standard collection tube (e.g. K2EDTA plasma collection tube) is performed at the clinic or draw station. Plasma or serum is separated and if necessary they can be re-centrifuged to ensure cell-free status. The emphasis is on preparing cell-free material to limit background contamination due to lysis of nucleated cells in the blood. While this has led to the use of specialized collections tubes (Streck, NE, USA) in the field of prenatal diagnostics, these have not been widely tested for colorectal cancer screening. Cleared plasma can be tested immediately, or stored frozen for a period of time.

Nucleic acid extraction
In this step, cell-free nucleic acids are extracted from the plasma sample. While a number of commercial methods have been developed for this purpose, it remains the Achilles heel of the process. Given the wide range in target concentration, and particularly the exceptionally low copy number expected for early cancers (in the single copy per mL range) [6], as well as the fragmented nature of cell-free DNA, the extraction methods must be designed to handle large samples (e.g. 3–4 mL of plasma), and be able to isolate fragmented DNA. The use of magnetic particles for purification coupled with modified binding and wash buffers designed to capture the full range of DNA fragments has simplified the extraction, and with the development of liquid handling platforms that can process larger volumes, this step is becoming automatable. While the reduction from 3.5 mL plasma to 100 µL of DNA eluate would raise concerns for PCR inhibition, for DNA methylation tests, it is possible to reduce the wash steps because the DNA is extensively purified in the bisulfite treatment process.

Bisulfite treatment
The bisulfite treatment process is required if the target is DNA methylation-based. Recent improvements in bisulfite conversion technology have simplified the treatment. The change to ammonium bisulfite allows for liquid reagents – a key attribute for kit development. In combination with elevated temperatures, bisulfite incubation time is reduced to less than 1 hour, enabling single shift turn-around times for tests. Furthermore, the reaction can be purified using a magnetic particle extraction that takes advantage of the same particles used for the initial DNA extraction. This process can also be automated on a standard liquid handling platform to improve throughput and quality.

Real-time PCR
For genetic (mutation)-based tests, the test can be performed immediately following initial DNA extraction, though it is important to increase the stringency of DNA washes to limit the potential for PCR inhibition. In the final steps, either genetic or epigenetic markers are measured by real-time PCR. For screening applications, the target concentration dictates the conditions and interpretation of the PCR reaction. For example, in the methylated Septin9 test, the final recovered bisulfite converted template DNA is split into three wells and run in three PCR reactions. Although the PCR reaction is run as a real-time assay, the test is essentially a qualitative end point test, since a well is called positive if a PCR curve occurs at any cycle during the course of the reaction. In addition, the results of the three reactions are combined to produce a final interpretation for a patient sample. For the CE-marked Epi proColon 2.0 product, the sample is called positive if two of three wells are positive. For the Ep proColon product undergoing US FDA PMA review, the sample is called positive if any of three wells are positive. This allows for a greater emphasis on a specific test parameter – for sensitivity (any well-positive) or test specificity (two out of three wells positive).

Summary
The use of genetic and epigenetic biomarkers for cancer screening is a field still in its infancy that has great opportunities for growth. Because these biomarkers can be used as indicators of disease, they also have diagnostic and prognostic potential that will be incorporated into the clinical-decision making process. For CRC screening, test kits are already available in Europe and other countries, and are currently under review by both the US and Chinese FDA organizations. In the US, LDTs are currently marketed, and together, all progress represents significant opportunities to generate positive momentum. The introduction of simple, blood-based screening would provide a viable alternative to patients refusing or avoiding current well established methods. The convenience factors of sample collection and processing by health professionals also avoids the challenges of faulty sampling, handling, and mailing associated with at-home self-collected tests. Finally, given the extensive collection of promising biomarkers on the horizon, mechanisms are needed now to expedite clinical utilization and validation to drive further improvements in test performance.

References
1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from: globocan.iarc.fr, accessed on 12/09/2014.
2. Kuipers EJ, Rösch T, Bretthauer M. Colorectal cancer screening – optimizing current strategies and new directions. Nat Rev Clin Oncol. 2013; 10: 130–142.
3. Brenner H, Stock C, Hoffmeister M. Effect of screening sigmoidoscopy and screening colonoscopy on colorectal cancer incidence and mortality: systematic review and meta-analysis of randomised controlled trials and observational studies. BMJ 2014; 348: g2467.
4. American Cancer Society. Colorectal Cancer Facts & Figures 2014-2016. Atlanta: American Cancer Society, 2014.
5. Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014; 370(14): 1287–1297.
6. Potter NT, Hurban P, White MN, Whitlock KD, Lofton-Day CE, Tetzner R, Koenig T, Quigley NB, Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma. Clin Chem. 2014; 60(9): 1183–1191.
7. National Colorectal Cancer Round Table. Tools & Resources – 80% by 2018. nccrt.org/about/80-percent-by-2018/
8. Gimeno García AZ. Factors influencing colorectal cancer screening participation. Gastroenterol Res Pract. 2012; 2012: 483417.
9. Toiyama Y, Okugawa Y, Goel A. DNA methylation and microRNA biomarkers for noninvasive detection of gastric and colorectal cancer. Biochem Biophys Res Commun. 2014; doi: 10.1016/j.bbrc.2014.08.001.
10. Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, Duesing KR, Buckley MJ, Dunne R, Beetson I, Rand KN, McEvoy A, Thomas ML, Baker RT, Wattchow DA, Young GP, Lockett TJ, Pedersen SK, Lapointe LC, Molloy PL. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer 2014; 14: 54.

The author
Theo deVos PhD
Epigenomics Inc.,
Seattle, WA 98107, USA
E-mail: theo.devos@epigenomics.com

Read More

The Scientist> The Nutshell> WHO: TB’s Toll Worse Than Thought

A new report from the World Health Organization finds that tuberculosis has infected hundreds of thousands more people around the world than was estimated a year ago.

By Bob Grant | October 22, 2014

 

Mycobacterium tuberculosis, the bug that causes TB
WIKIMEDIA, NIAID

   Tuberculosis (TB) is causing more infections and deaths the world over than previous estimates indicated, according to a new survey released by the World Health Organization (WHO) today (October 22). The WHO’s “Global Tuberculosis Report 2014” stated that in 2013 there were 9 million new cases of TB reported in the more than 200 countries that account for more than 99 percent of the world’s TB cases.

The number of reported cases this year is 400,000 more than the WHO estimated in last year’s report, but the increased numbers may indicate improvements in diagnosis and data reporting as well as unchecked spread of the disease. “There has been some real progress, particularly in Asia, but the overall situation remains catastrophic,” Richard Chaisson, director of the Johns Hopkins Center for Tuberculosis Research in Baltimore, Maryland, told ScienceInsider. “Improvements in some countries are offset by disastrous situations in others, with MDR [multidrug-resistant] TB, HIV-related TB, and continued high rates of

missed diagnoses and deaths. The situation in Africa is particularly horrific, with TB killing more young people than any other cause.”

 

TB kills hundreds of thousands of people every year—an estimated 1.5 million people died from the disease in 2013, according to the WHO report—second only to HIV among infectious diseases.

Some infectious disease activists are criticizing the WHO report for being overly optimistic. “On the HIV side, we're doing stuff almost twice as fast in [reducing] deaths and multiple times as fast in incidence, even though TB is curable and HIV is not,” Mark Harrington, executive director of the New York City–based Treatment Action Group, which lobbies for stronger efforts to address both HIV and TB, told ScienceInsider.

Tags

WHO, tuberculosis, TB, statistics, infectious disease, global health, epidemiology, drug resistance and disease/medicine

 

 

Read More

The Nutshell: Tumor Exosomes Make microRNAs

Tumor Exosomes Make microRNAs

Cellular blebs shed by tumor cells can process short stretches of RNA that go on to induce tumor formation in neighboring cells.

By Kerry Grens | October 27, 2014

 

WIKIMEDIA, NATIONAL CANCER INSTITUTE

Cells give off little membrane-bound bubbles called exosomes, which are known to contain microRNAs (miRNAs)—short stretches of RNA that can interfere with protein production. Exosomes have also been associated with cancer metastasis. According to a  study published in Cell last week (October 23), tumor-linked exosomes can produce their own miRNAs from starter material, and these miRNAs can enter neighboring cells and incite them to form tumors.

 

“Collectively, this study unravels the possible role cancer exosomes play in inducing an oncogenic ‘field effect’ that further subjugates adjacent normal cells to participate in cancer development and progression,” Raghu Kalluri, a cancer biologist at MD Anderson Cancer Center, and his coauthors wrote in their report.

 

Comparing exosomes derived from normal cells to those derived from breast cancer cells, Kalluri’s

team found the proteins responsible for chopping up miRNA precursors into their mature form present only in the cancer exosomes. When normal cells were implanted into a mouse alongside cancer exosomes, those normal cells formed tumors. However, blocking an miRNA-processing protein called Dicer in the cancer exosomes obliterated tumor formation, “suggesting that miRNA biogenesis in exosomes contributes to cancer progression,” the authors wrote.

 

“It’s amazing—these vesicles were considered garbage cans,” Khalid Al-Nedawi, a cancer researcher at McMaster University in Hamilton, Canada, told Nature News. “This paper really brings us closer to harnessing the potential of these tiny vesicles.”

 

Tags: miRNA, microRNA, metastasis, exosomes, cell & molecular biology and cancer

 

 

Read More

Immune cells in experimental acute kidney injury

Nature Reviews Nephrology:

 

The immune system has a vital role in the renal response to acute kidney injury (AKI). In this Review, Hye Ryoun Jang and Hamid Rabb describe current understanding of the function of the innate and adaptive immune systems in the early and late injury phases of ischaemic and nephrotoxic AKI, and describe the influence of immune cells on recovery and long-term outcome following AKI. 

 

Abstract:

 

Acute kidney injury (AKI) prolongs hospital stay and increases mortality in various clinical settings. Ischaemia–reperfusion injury (IRI), nephrotoxic agents and infection leading to sepsis are among the major causes of AKI. Inflammatory responses substantially contribute to the overall renal damage in AKI. Both innate and adaptive immune systems are involved in the inflammatory process occurring in post-ischaemic AKI. Proinflammatory damage-associated molecular patterns, hypoxia-inducible factors, adhesion molecules, dysfunction of the renal vascular endothelium, chemokines, cytokines and Toll-like receptors are involved in the activation and recruitment of immune cells into injured kidneys. Immune cells of both the innate and adaptive immune systems, such as neutrophils, dendritic cells, macrophages and lymphocytes contribute to the pathogenesis of renal injury after IRI, and some of their subpopulations also participate in the repair process. These immune cells are also involved in the pathogenesis of nephrotoxic AKI. Experimental studies of immune cells in AKI have resulted in improved understanding of the immune mechanisms underlying AKI and will be the foundation for development of novel diagnostic and therapeutic targets. This Review describes what is currently known about the function of the immune system in the pathogenesis and repair of ischaemic and nephrotoxic AKI

 

Key points:

• Various components of the innate and adaptive immune systems are implicated in the pathogenesis and repair of acute kidney injury (AKI)

• The roles of individual immune cell types have been most thoroughly investigated in models of ischaemic AKI

• Various immune cells traffic into the post-ischaemic kidney and show changes in phenotypes and numbers depending on the time course after establishment of ischaemic AKI

• The roles of macrophages, renal dendritic cells and T regulatory cells differ according to the pathogenesis of AKI

• Although numerous studies in animal models of AKI show therapeutic potential for modulating immune cells, big hurdles must be overcome before applying these findings to patients

• Functions and interactions of specific immune cell types and humoral factors in AKI differ between human disease and animal models, and depend on the type and stage of injury

Introduction

 

Despite remarkable advances in modern medicine, acute kidney injury (AKI) still remains a challenging condition that lacks specific tools for its early diagnosis and treatment. AKI worsens the overall clinical course of affected patients by causing uraemia, acid–base or electrolyte disturbances, and volume overload. The incidence of AKI has been reported to be as high as 5% of hospitalized patients or 30% of critically ill patients.¹ The risk of chronic kidney disease and end-stage renal disease is substantially increased in patients with AKI.² Most patients with AKI are diagnosed when injury is already established and, therefore, only conservative treatment including fluid therapy and dialysis is available. To improve the clinical outcome of AKI, novel diagnostic and therapeutic strategies need to be developed. Understanding the pathophysiology of AKI is, therefore, the cornerstone of exploration of novel diagnostic and therapeutic strategies.

 

Experimental models of AKI can be divided into several categories depending on the induction method (Figure 1). In models of septic AKI, the initial immune response against foreign antigens and innate triggers causes a complex secondary inflammatory response that facilitates renal injury.³ Non-septic and septic AKI are known to have very different pathophysiological features. Septic AKI is a systemic manifestation of sepsis following exposure to foreign antigens such as bacteria or viruses; detailed discussion of septic AKI is beyond the scope of this review.

 

Immune mechanisms were not expected to have an important role in models of aseptic AKI, but numerous studies conducted over the past two decades have revealed that inflammatory processes mediated by the immune system are crucial in mediating renal injury.³ Immune mechanisms involved in the pathogenesis of renal injury have been studied most extensively in models of ischaemic AKI employing cold or warm ischaemia. Both types of ischaemia occur during organ transplantation; cold ischaemia starts when the organ is cooled with cold perfusion solution after procurement, and lasts until the temperature of the organ reaches the physiologic temperature. Thereafter, warm ischaemia begins, and ends when perfusion is restored after completion of surgical anastomosis. Thus, two distinct periods of warm ischaemia occur in the transplantation setting—during organ retrieval and implantation.⁴ Interestingly, the nephrotoxicity induced by cisplatin, a chemotherapeutic agent, has many pathophysiological features that overlap with those of ischaemia–reperfusion injury (IRI).

 

Both innate and adaptive immune systems are directly involved in the pathogenesis of ischaemic AKI. Various cellular and humoral immune system components contribute to AKI, some of which are also thought to be involved in the repair process following IRI.^{5, 6} The healthy kidney produces hormones that influence the immune system, such as vitamin D (calcitriol) and erythropoietin,⁷ and the renal tubular epithelium expresses Toll-like receptors (TLRs), which critically contribute to activation of the complement system and recruitment of immune cells in response to inflammatory stimuli.^{8, 9} Several types of resident immune cells, such as dendritic cells, macrophages, mast cells and lymphocytes are homeostatically maintained in the normal kidney, although these cells constitute a small population.^{10, 11, 12, 13} Under normal conditions, the renal mononuclear phagocytes mainly comprise macrophages located in the renal medulla and capsule and renal dendritic cells found in the tubulointerstitium.^{10, 11, 14} In mice, renal dendritic cells show a specific CD11c⁺CD11b⁺EMR1(F4/80)⁺CX₃CR1 (CX₃C-chemokine receptor)⁺CD8⁻CD205⁻ phenotype, and have a similar transcriptome as dendritic cells residing in other nonlymphoid tissues.^{15, 16} Dendritic cells are recruited to the kidney by a CX₃CR1–CX₃CL1 (CX₃C-chemokine ligand 1, also known as fractalkine) chemokine pair,¹⁷ and have an important role in local injury or infection. Dendritic cells not only function as a potent source of other factors, such as neutrophil-recruiting chemokines and cytokines,^{12, 18} but also present antigens to T cells. Intrarenal macrophages exert homeostatic functions by phagocytosis of antigens in the kidney and undergo phenotypic changes that enable them to participate in both inflammatory and anti-inflammatory processes.¹⁴ Both dendritic cells and macrophages contribute substantially to homeostasis and regulation of immune responses (as resident renal mononuclear phagocytes) in the normal kidney. Mast cells also reside in the tubulointerstitium and mediate pathogenic processes in crescentic and other forms of glomerulonephritis. However, the exact roles of dendritic cells, macrophages and mast cells in the normal kidney are yet to be elucidated.^{19, 20, 21} Lymphocytes, including both T cells and B cells, have been found in normal mouse kidneys even after extensive exsanguination and perfusion.²² Intrarenal resident T cells show distinctly different phenotypes from T cells in spleen and blood; those from normal mouse kidneys contain an increased percentage of CD3⁺CD4⁻CD8⁻ double-negative T cells. Intrarenal T cells also show a high proportion of activated, effector and memory phenotypes, whereas a small percentage of regulatory T cells and natural killer (NK) T cells exist in perfused and exsanguinated mouse kidney.²²

 

In this Review, we describe how immune cells participate in the pathogenesis of AKI, focusing on ischaemic and nephrotoxic AKI. Immune system function in septic AKI is only outlined in this article, because the pathophysiology of septic AKI includes both immune responses to various foreign antigens and secondary systemic inflammatory responses, which are distinctly different to the immune responses that occur in aseptic AKI.

 

Figure 1: Experimental models of AKI.

Models of AKI can be broadly categorized according to whether foreign antigens are involved (aseptic or septic AKI). Each category can be subdivided according to the method used to induce AKI. Ischaemic AKI is induced by ischaemia–reperfusion injury and by the type of ischaemia (warm or cold). Nephrotoxic AKI is induced by nephrotoxic agents, such as cisplatin. Abbreviation: AKI, acute kidney injury.

 

Figure 2: Major effector cells of both innate and adaptive immune systems contribute to the establishment of renal injury in ischaemic AKI.

An immune response is initiated in post-ischaemic kidneys by resident immune cells and is potentiated by a rapid influx of immune cells through the disrupted endothelium. TLRs, adhesion molecules and DAMPs released from dying cells facilitate the recruitment and activation of various immune cells including neutrophils, macrophages, dendritic cells, NK cells, T cells and B cells during the early injury phase. Activation of the complement system and increased production of proinflammatory cytokines and chemokines are important promoters of leucocyte infiltration into the post-ischaemic kidney. Major effector cells of the innate immune system, such as macrophages, dendritic cells and NK cells are involved in the pathogenesis of renal injury after IRI. T cells, the major effector cells of the adaptive immune system, also substantially contribute to the development of renal injury from the early to late injury phase. Plasma cells seem to participate in the tubular damage process during the late injury phase. Abbreviations: AKI, acute kidney injury; DAMPs, damage-associated molecular patterns; HIF, hypoxia-inducible factor; IRI, ischaemia–reperfusion injury; NK, natural killer; TLR, Toll-like receptor; T_REG cell, regulatory T cell. Modified with permission from Elsevier © Jang, H. R. & Rabb, H. The innate immune response in ischemic acute kidney injury. Clin. Immunol. 130, 41–50 (2009).

Figure 3: Immune modulation during the repair phase of ischaemic AKI is a key factor in determining the outcome of AKI.

T_REG cells, B cells and macrophages have substantial roles in determining whether repair results in tubular regeneration or atrophy and interstitial fibrosis. T_REG cells and M2 macrophages have important roles in tubular regeneration, whereas B cells enhance tubular atrophy and suppress tubular regeneration. Humoral factors, such as proinflammatory or anti-inflammatory cytokines and chemokines, also change the intrarenal microenvironment and affect phenotype switching of macrophages. The exact mechanisms by which these immune processes regulate tubular atrophy or regeneration are not yet known. Abbreviations: AKI, acute kidney injury; IL-10, interleukin-10; TGF-β, transforming growth factor β; T_REG cells, regulatory T cells. Modified with permission from Elsevier © Jang, H. R. & Rabb, H. The innate immune response in ischemic acute kidney injury. Clin. Immunol. 130, 41–50 (2009).

 

Figure 4: Important immune cells in each phase of renal IRI.

 Neutrophils and NK T cells infiltrate the post-ischaemic kidney in the early injury phase and contribute to initiation of the inflammatory cascade. NK cells also contribute to renal tissue injury in the early injury phase. Renal dendritic cells increase in number and are activated to mediate inflammation from the early to late injury phase. Macrophages have diverse roles throughout the pathogenesis of renal IRI. In the injury phase, M1 macrophages contribute to inflammation and tissue injury, whereas M2 macrophages exert anti-inflammatory functions in post-ischaemic kidneys and facilitate renal tubular regeneration during the recovery phase. T cells also show dynamic changes in number and phenotype depending on the phase of renal IRI. CD4⁺ T cells have a substantial role in inducing renal tissue damage in the early injury phase. T_REG cells increase in the late injury phase and facilitate tubular regeneration in the recovery phase. B cells are activated and differentiate in the injury phase, and limit tubular regeneration in the recovery phase. Abbreviations: DAMPs, damage-associated molecular patterns; IRI, ischaemia–reperfusion injury; NK, natural killer; TLR, Toll-like receptor; T_REG cells, regulatory T cells.

Hye Ryoun Jang^1, & Hamid Rabb^2, 

 

Published online 21 October 2014

                    

 

Read More

Male infertility: a public health issue caused by sexually transmitted pathogens

Nature Reviews Urology:

 

Sexually transmitted diseases caused by bacteria, viruses and protozoa can affect the male genital tract. Gimenes and colleagues discuss the evidence for effects of sexually transmitted pathogens on semen, sperm and male infertility.

 

Fabrícia Gimenes,^1, Raquel P. Souza,^1, Jaqueline C. Bento,^3, Jorge J. V. Teixeira,^2, Silvya S. Maria-Engler,^4, Marcelo G. Bonini^5, & Marcia E. L. Consolaro^1,

 

Published online: 21 October 2014

 

Abstract:

 

Sexually transmitted diseases (STDs) are caused by several pathogens, including bacteria, viruses and protozoa, and can induce male infertility through multiple pathophysiological mechanisms. Additionally, horizontal transmission of STD pathogens to sexual partners or vertical transmission to fetuses and neonates is possible. Chlamydia trachomatis, Ureaplasma spp., human papillomavirus, hepatitis B and hepatitis C viruses, HIV-1 and human cytomegalovirus have all been detected in semen from symptomatic and asymptomatic men with testicular, accessory gland and urethral infections. These pathogens are associated with poor sperm quality and decreased sperm concentration and motility. However, the effects of these STD agents on semen quality are unclear, as are the effects of herpes simplex virus type 1 and type 2, Neisseria gonorrhoeae, Mycoplasma spp., Treponema pallidum and Trichomonas vaginalis, because few studies have evaluated the influence of these pathogens on male infertility. Chronic or inadequately treated infections seem to be more relevant to infertility than acute infections are, although in many cases the exact aetiological agents remain unknown.

 

Key points 

• Sexually transmitted diseases (STDs) can induce male infertility through multiple pathophysiological mechanisms
• Several STD-causing agents, including bacteria, viruses and protozoa have been detected in semen from symptomatic and asymptomatic males, and can be transmitted through natural intercourse or insemination
• STD pathogens can affect sperm parameters and functions, particularly when testicular, accessory gland and urethral infections localize the disease agents in proximity to semen
• Several highly sensitive and specific molecular methods are now available to explore the relationship between infertility and infections of semen with STD pathogens
• Chlamydia trachomatis, Ureaplasma spp., human papillomavirus, hepatitis B and C viruses, HIV-1 and human cytomegalovirus are associated with reduced sperm quality, concentration

Introduction

 

Sexually transmitted diseases (STDs) are caused by microorganisms that colonize the female and male genital tracts, often causing only mild symptoms.¹ STDs affect health and cause social and economic problems worldwide.^{2, 3} Despite the development of antibiotics and vaccines, and the existence of disease prevention and control programmes, these pathogens remain important causes of acute and chronic disease.¹

 

In 2000, the WHO recognized the role of genital tract infections in human infertility.⁴ STDs in men cause genital injury, infections of semen, prostatitis, urethritis, epididymitis and orchitis.⁵ Several studies have reported that infertile men are affected by semen infection,⁶ mainly resulting from testicular, accessory gland and urethral infections. Semen infection also has possible involvement in pregnancy complications and, via maternal infection, in transmission of pathogenic agents to the fetus or neonate.¹ STD-causing microorganisms detected in semen have been associated with poor sperm quality,⁷ decreases in sperm concentration and motility^{8, 9} and changes in other semen parameters relevant to fertility, including sperm velocity, morphology and viability, as well as seminal volume, pH, viscosity and biochemical composition.^{7, 8, 9}

 

Several STD pathogens have been detected in semen from asymptomatic and symptomatic men (Figure 1 and Table 1).^{10, 11, 12, 13, 14} Chronic or inadequately treated infections seem to have greater association with infertility than acute infections do, although in many cases the exact aetiological agents resulting in infertility remain unknown.¹ Undiagnosed, asymptomatic infections could have important implications for individual and public health.¹⁵

 

Figure 1: Major sexually transmitted disease pathogens detected in semen.

Inflammatory processes triggered in the male genital tract (MGT) by some STDs can lead to deterioration of spermatogenesis and obstruction of the seminal tract,^{16, 17} which can worsen the characteristics of semen (Figure 2 and Table 2).¹⁸ Abortive apoptosis is an important part of the control of spermatogenesis, and has been observed in spermatogonia, spermatocytes, spermatids and ejaculated spermatozoa.^{18, 19, 20} However, sperm apoptosis can also be associated with inflammatory conditions and oxidative stress that occur in response to infection, resulting in impaired motility and reduced capacity of spermatozoa for fertilization.^{18, 19, 20, 21, 22}

Figure 2: Sexually transmitted disease loci in the male genital tract and their relation to infertility.

The demonstration of a microorganism in the male genital tract and seminal fluid can be a sign of an infection with pathological sequelae. The inflammatory processes triggered by infection with some pathogens can lead to deterioration of spermatogenesis and obstruction of the seminal tract.

Over the past three decades, diagnostics for STDs have depended on traditional methods, such as bacterial culture, enzyme immunoassays, fluorescent antibody staining and microscopy. In recent years, several highly sensitive and specific PCR-based diagnostic methods have become available, enabling better understanding of the relationship between infertility and infections of semen with STD pathogens.²³

Figure 3: Interaction of sexually transmitted disease pathogens with spermatogenic cells and spermatozoa.

a | Schematic diagram and representative photomicrographs (with Leishman staining) of spermatogenesis, indicating pathogens that are known to attach and internalize in spermatogenic cells. b | Photomicrograph indicating sperm death by staining (red) with eosin-nigrosin, and schematic illustration of a spermatozoon showing sites of attachment and internalization of sexually transmitted disease pathogens. Internalization in the sperm head can cause DNA damage.

 

 

Read More