Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells.

Mitochondrial damage and the resultant oxidative stress are associated with neurodegenerative diseases, ageing, and cancer. However, the triggers of mitochondrial damage remain unclear. We previously reported that cell-free chromatin particles (cfChPs) released from the billions of cells that die in the body every day can readily enter healthy cells and damage their DNA. Here, we show that cfChPs isolated from the sera of healthy individuals, when applied to NIH3T3 mouse fibroblast cells, cause physical damage to mitochondrial DNA (mtDNA). cfChPs also induce ultrastructural changes, increase mitochondrial mass, alter mitochondrial shape, upregulate mitochondrial outer membrane protein translocase of the outer membrane 20, and change mitochondrial membrane potential. Furthermore, a marked increase was observed in mitochondrial superoxide (ROS) production, as detected by MitoSOX Red, and intracellular superoxide dismutase-1 activation. ROS production was also activated when a conditioned medium containing cfChPs released from hypoxia-induced dying NIH3T3 cells was applied to healthy NIH3T3 cells. ROS activation was significantly reduced when the conditioned medium was pre-treated with three different cfChP-deactivating agents: anti-histone antibody-complexed nanoparticles, DNase I, and the novel pro-oxidant combination of the nutraceuticals resveratrol and copper. Given that 1 × 109-1 × 1012 cells die in the body every day, we hypothesise that cfChPs from dying cells are the major physiological triggers for mtDNA damage and ROS production. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions linked to oxidative stress.

Cell-free chromatin particles released from dying cancer cells activate immune checkpoints in human lymphocytes: implications for cancer therapy.

Immune checkpoint blockade is the exciting breakthrough in cancer, but how immune checkpoints are activated is unknown. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in blood of cancer patients, or those that are released locally from dying cancer cells, are readily internalized by healthy cells with biological consequences. Here we report that treatment of human lymphocytes with cfChPs isolated from sera of cancer patients led to marked activation of the immune checkpoints PD-1, CTLA-4, LAG-3, NKG2A, and TIM-3. This finding was corroborated in vivo in splenocytes of mice when cfChPs were injected intravenously. Significant upregulation of immune checkpoint was also observed when isolated lymphocytes were exposed to conditioned medium containing cfChPs released from hypoxia-induced dying HeLa cells. Immune checkpoint activation could be down-regulated by pre-treating the conditioned media with three different cfChPs deactivating agents. Down-regulation of immune checkpoints by cfChPs deactivating agents may herald a novel form of immunotherapy of cancer.

The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.

A New Perspective on the Origin of DNA Double-Strand Breaks and Its Implications for Ageing.

It is estimated that 10-50 DNA double-strand breaks (DSBs) occur in a nucleated human cell per cell cycle. We reviewed the present state of knowledge and hypothesized that the currently accepted mechanisms cannot explain such high frequency of DSBs occurring daily under normal physiological conditions. We propose an alternative model that implicates illegitimate genomic integration into healthy cells of cell-free chromatin (cfCh) particles released from the billions of cells that die in the body every day. Repeated genomic integration of cfCh may have catastrophic consequences for the cell, such as DSBs, their faulty repair by nonhomologous end joining (NHEJ) followed by apoptosis with release of more cfCh which would integrate into genomes of surrounding cells. This can creates a vicious cycle of cfCh integration, DSBs, NHEJ, and more apoptosis, thereby providing a potential explanation as to why so many billions of cells die in the body on a daily basis. We also recount the recent observation that cfCh integration and the resulting DSBs activate inflammatory cytokines. This leads us to propose that concurrent DSBs and induction of inflammation occurring throughout life may be the underlying cause of ageing, degenerative disorders, and cancer. Finally, we discuss the prospect that agents that can inactivate/degrade cfCh may hold the key to making healthy ageing a realizable goal.

Cytokine Storm as a Cellular Response to dsDNA Breaks: A New Proposal.

Pathogenesis of cytokine storm is poorly understood. In this article we propose a new mechanism and suggest innovative therapeutic avenues for its prevention. We have reported that particles of cell-free chromatin (cfCh) that are released from the billions of cells that die in the body everyday can illegitimately integrate into genomes of healthy cells to trigger dsDNA breaks. The latter leads to apoptosis and/or intense activation of inflammatory cytokines in the affected cells. We hypothesise that a similar phenomenon of dsDNA breaks and inflammation is involved in cytokine storm. The abundant cfCh particles that are released from dying host cells following viral/microbial invasion initiate a cascading effect of more cell death resulting in a vicious cycle of further DNA damage, apoptosis and hyper-inflammation which culminate in cytokine storm. We propose that this unrelenting vicious cycle of cellular DNA damage and cytokine storm may be the underlying cause of high mortality from severe COVID-19. We discuss results of our preclinical studies wherein we have shown that endotoxin induced cytokine storm in mice can be reversed by three different agents that have the ability to inactivate cfCh. These agents may be worthy of investigation in clinical trials to reduce mortality from COVID-19.

Spectrum of Inborn errors of immunity in a cohort of 90 patients presenting with complications to BCG vaccination in India.

World Health Organisation recommends the practice of BCG vaccination at birth in countries which have a high incidence of tuberculosis and/or high leprosy burden. The BCG vaccination is considered safe for a competent immune system. However, in children with weakened immune systems cause of which can be primary or secondary, the vaccine may lead to side effects which can be localized or disseminated. In this study, we report a spectrum of inborn errors of immunity (IEI) commonly referred to as primary immunodeficiency disorders (PIDs) diagnosed in a large cohort of patients presenting with complications to BCG vaccination from India. Retrospective data analysis of patients referred to ICMR- National Institute of Immunohematology (ICMR-NIIH) for IEI workup between 2007 and 2019 was done. IEI was identified in n = 52/90 (57.7%) patients presenting with BCG complications. Of these, n = 13(14.4%) patients were diagnosed with severe combined immune deficiency, n = 15(16.7%) with chronic granulomatous disease, n = 19(21.1%) with Inborn errors of IFN-γ immunity, n = 4(4.4%) with Combined immunodeficiency and n = 1(1.1%) with Leucocyte Adhesion Deficiency type1. Majority of cases with BCGosis (88%) had an underlying IEI. This study strongly highlights the need for evaluation of patients with BCG complications for underlying IEI. While disseminated BCGosis strongly predicts underlying IEI, even localized persistent adenitis may be a warning sign of underlying IEI. It is also strongly recommended to record a family history of previous sibling death prior to administration of this live vaccine and deferring live vaccine till the diagnosis of IEI is ruled out in cases with a positive family history.

Prenatal Diagnosis for Primary Immunodeficiency Disorders-An Overview of the Indian Scenario.

Prenatal Diagnosis (PND) forms an important part of primary preventive management for families having a child affected with primary immunodeficiency. Although individually sparse, collectively this group of genetic disorders represents a significant burden of disease. This paper discusses the prenatal services available for affected families at various centers across the country and the challenges and ethical considerations associated with genetic counseling. Mutation detection in the index case and analysis of chorionic villous sampling or amniocentesis remain the preferred procedures for PND and phenotypic analysis of cordocentesis sample is reserved for families with well-characterized index case seeking PND in the latter part of the second trimester of pregnancy. A total of 112 families were provided PND services in the last decade and the presence of an affected fetus was confirmed in 32 families. Post-test genetic counseling enabled the affected families to make an informed decision about the current pregnancy.

Impaired NK cell activation during acute dengue virus infection: A contributing factor to disease severity.

Dengue viral (DENV) infection has a broad clinical spectrum ranging from classical febrile illness to life-threatening disease. Literature suggests that spectrum of illness could be due to differences in innate immune-responses; however, the knowledge is still at infancy. Amongst the various cells involved in innate immune responses, NK cells play a central role, particularly in anti-viral immunity. Thus in this study we have evaluated the role of NK-cells during acute-DENV infection and its influence on severity of disease, by analyzing activation, cytotoxic receptors, cytolytic granule contents and degranulation markers on NK-cells during different stages of infection. Based on the clinical manifestations and severity of the disease, DENV patients were classified into patients with dengue without warning signs (DF), dengue with warning signs (DFWS) and severe dengue (SD) patients. During acute-DENV infection, though there was no alteration in frequency of NK-cells, significant increase in frequency of CD56bright subset in DF patients (p < 0.05) was observed, while it remained unaltered in SD patients. We also found that, CD56dim NK-cell subset of DF patients had elevated CD69 expression, granzyme B and intracellular IFN-γ levels compared to SD patients (p < 0.05). Amongst the NK-cell cytotoxicity receptor (NCR), NKp30 receptor was significantly elevated in DF patients (p < 0.05), however in SD patients it was comparable to healthy controls. This receptor is essential for dendritic cells-NK-cells crosstalk for initiating adaptive immune response. IL-15 is known to induce NKp30 expression, which was also seen to be elevated in DF patients (p < 0.05) but unaltered in SD patients. In SD patients, even post-6 days of infection i.e. during recovery phase, CD69 and NKp30 expression did not raise, suggesting impaired NK-cell response in these patients. To summarize, our study reports, that efficient NK cell response during acute phase of DENV infection is crucial for preventing severity of the disease. This study helps in understanding the dynamics of NK cell response in immunopathogenesis of DENV infection; which is crucial for development of efficacious therapeutics as well as vaccine.

Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.

Primary immunodeficiency diseases (PID) are a clinically and immunologically heterogeneous group of disorders of immune system. Diagnosis of these disorders is often challenging and requires identification of underlying genetic defects, complemented by a comprehensive evaluation of immune system. Flow cytometry, with its advances in the last few decades, has emerged as an indispensable tool for enumeration as well as characterization of immune cells. Flow cytometric evaluation of the immune system not only provides clues to underlying genetic defects in certain PIDs and helps in functional validation of novel genetic defects, but is also useful in monitoring immune responses following specific therapies. India has witnessed significant progress in the field of flow cytometry as well as PID over last one decade. Currently, there are seven Federation of Primary Immunodeficiency Diseases (FPID) recognized centers across India, including two Indian Council of Medical research (ICMR) funded centers of excellence for diagnosis, and management of PIDs. These centers offer comprehensive care for PIDs including flow cytometry based evaluation. The key question which always remains is how one selects from the wide array of flow cytometry based tests available, and whether all these tests should be performed before or after the identification of genetic defects. This becomes crucial, especially when resources are limited and patients have to pay for the investigations. In this review, we will share some of our experiences based on evaluation of a large cohort of hemophagocytic lymphohistiocytosis, severe combined immunodeficiency, and chronic granulomatous disease, and the lessons learned for optimum use of this powerful technology for diagnosis of these disorders.

Natural Killer Cell Degranulation Defect: A Cause for Impaired NK-Cell Cytotoxicity and Hyperinflammation in Fanconi Anemia Patients.

Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.

Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function.

Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca(2+)-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p < 0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

Current Updates on Classification, Diagnosis and Treatment of Hemophagocytic Lymphohistiocytosis (HLH).

Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyperinflammatory syndrome characterized by excessive activation of macrophages and T cells resulting from defective cytotoxicity. Severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes (macrophages) secreting high amounts of inflammatory cytokines threatens the life of the patient and may lead to death unless arrested by appropriate treatment. HLH can be caused either by certain underlying genetic diseases (familial HLH), or may also occur due to particular triggers in patients with no known inherited disorder (acquired HLH). Due to life threatening nature of the disease, early diagnosis and initiation of immunosuppressive therapy is extremely important. HLH diagnosis is based on constellation of clinical manifestations and laboratory parameters which often overlap with those of severe infection or sepsis. Identification of patients with familial HLH and their underlying genetic defects requires specialized laboratory tests and is important for predicting relapses and planning early therapeutic hematopoietic stem cell transplantation (HSCT). A high suspicion and thorough clinical, immunological and genetic work-up is required for diagnosis of HLH. Prompt initiation of adequate treatment is essential for the survival. Substantial progress has been made in exploring the complex cause and pathophysiology of HLH and also in management of HLH patients.