Negative impact of Interleukin-9 on synovial regulatory T cells in rheumatoid arthritis.

In Rheumatoid Arthritis (RA), regulatory T cells (Tregs) have been found to be enriched in the synovial fluid. Despite their accumulation, they are unable to suppress synovial inflammation. Recently, we showed the synovial enrichment of interleukin-9 (IL-9) producing helper T cells and its positive correlation with disease activity. Therefore, we investigated the impact of IL-9 on synovial Tregs in RA. Here, we confirmed high synovial Tregs in RA patients, however these cells were functionally impaired in terms of suppressive cytokine production (IL-10 and TGF-ß). Abrogating IL-9/ IL-9 receptor interaction could restore the suppressive cytokine production of synovial Tregs and reduce the synovial inflammatory T cells producing IFN-γ, TNF-α, IL-17. However, blocking these inflammatory cytokines failed to show any effect on IL-9 producing T cells, highlighting IL-9's hierarchy in the inflammatory network. Thus, we propose that blocking IL-9 might dampen synovial inflammation by restoring Tregs function and inhibiting inflammatory T cells.

Interleukin-9 rescues class switching of Memory B cells derived from Common variable immunodeficiency patients.

Impaired class switch memory (CSM) B cell formation is the hallmark of common variable immunodeficiency (CVID). Various T cell abnormalities have been observed in CVID patients indicating inadequate T-cell help to B cells. A major setback in understanding its pathogenesis is due to diverse clinical presentation. Therefore, we performed extensive immunological investigation in a cohort of CVID patients with similar clinical findings in order to unravel the T cell dysfunction and its influence on the defective humoral immune response. All recruited CVID patients exhibited B cells in the normal range, but reduced CSM B cells. However, patients showed reduced T cell proliferation, reduced level of serum Interleukin-9 (IL-9) and frequency of IL-9 expressing CD4 (Th-9) cells. IL-9 supplementation along with CD40 engagement was effective in inducing in vitro CSM B cells formation in CVID patients. Thus, IL-9 supplementation has the potential to restore impaired CSM B cell formation in CVID.

T-cell signature cytokines distinguish pulmonary sarcoidosis from pulmonary tuberculosis.

Sarcoidosis is a systemic inflammatory disorder characterized by tissue infiltration due to mononuclear phagocytes and lymphocytes and associated noncaseating granuloma formation. Pulmonary sarcoidosis (PS) shares a number of clinical, radiological, and histopathological characteristics with that of pulmonary tuberculosis (PTB). Due to this, clinicians face issues in differentiating between PS and PTB in a substantial number of cases. There is a lack of any specific biomarker that can diagnose PS distinctively from PTB. We compared T-cell-based signature cytokines in patients with PS and PTB. In this study, we proposed a serum biomarker panel consisting of cytokines from cells: T helper (Th) 1 [interferon-gamma (IFN-γ); tumor necrosis factor-alpha (TNF-α)], Th9 [interleukin (IL)-9], Th17 [IL-17], and T regulatory (Treg) [IL-10; transforming growth factor-beta (TGF-ß)]. We performed the principal component analysis that demonstrated that our serum cytokine panel has a significant predictive ability to differentiate PS from PTB. Our results could aid clinicians to improve the diagnostic workflow for patients with PS in TB endemic settings where the diagnosis between PS and PTB is often ambiguous.

CD40 induces selective routing of Ras isoforms to subcellular compartments.

Ras GTPases are central to cellular signaling and oncogenesis. The three loci of the Ras gene encode for four protein isoforms namely Harvey-Ras (H-Ras), Kirsten-Ras (K-Ras 4A and 4B), and Neuroblastoma-Ras (N-Ras) which share ~ 80% sequence similarity and used to be considered functionally redundant. The small molecule inhibitors of Ras lack specificity for the isoforms leading to widespread toxicity in Ras-targeted therapeutics. Ras isoforms' tissue-specific expression and selective association with carcinogenesis, embryonic development, and infection suggested their non-redundancy. We show that CD40, an antigen-presenting cell (APC)-expressed immune receptor, induces selective relocation of H-Ras, K-Ras, and N-Ras to the Plasma membrane (PM) lipid rafts, mitochondria, endoplasmic reticulum (ER), but not to the Golgi complex (GC). The two palmitoylated Ras isoforms-H-Ras and N-Ras-have a similar pattern of colocalization into the lipid-rich raft microdomain of the PM at early time points when compared to non-palmitoylated K-Ras (4B) with polylysine residues. CD40-induced trafficking of H-Ras and K-Ras to mitochondria and ER was found to be similar but different from that of N-Ras. Trafficking of all the Ras isoforms to the GC was independent of CD40 stimulation. The receptor-driven trafficking and spatial segregation of H-Ras, K-Ras, and N-Ras imply isoform-specific subcellular signaling platforms for the functional non-redundancy of Ras isoforms. PDB structures have been modified to illustrate various signaling proteins.

Mechanistic insight of interleukin-9 induced osteoclastogenesis.

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.

Accelerating UN Sustainable Development Goals with AI-Driven Technologies: A Systematic Literature Review of Women's Healthcare.

In this paper, we critically examine if the contributions of artificial intelligence (AI) in healthcare adequately represent the realm of women's healthcare. This would be relevant for achieving and accelerating the gender equality and health sustainability goals (SDGs) defined by the United Nations. Following a systematic literature review (SLR), we examine if AI applications in health and biomedicine adequately represent women's health in the larger scheme of healthcare provision. Our findings are divided into clusters based on thematic markers for women's health that are commensurate with the hypotheses that AI-driven technologies in women's health still remain underrepresented, but that emphasis on its future deployment can increase efficiency in informed health choices and be particularly accessible to women in small or underrepresented communities. Contemporaneously, these findings can assist and influence the shape of governmental policies, accessibility, and the regulatory environment in achieving the SDGs. On a larger scale, in the near future, we will extend the extant literature on applications of AI-driven technologies in health SDGs and set the agenda for future research.

Are High-Altitude Residents More Susceptible to Covid-19 in India? Findings and Potential Implications for Research and Policy.

In this paper, we study the incidence of COVID-19 and the associated fatality with altitude using high frequency, district level data from India. To understand the implications of the nationwide lockdown after the outbreak, we use data for about four months- two from the lockdown period starting from March 25 till May 31, 2020 and about two months after unlocking was initiated (June 1-July 26, 2020). The multivariate regression result indicates slower growth in average rate of infection during the lockdown period in hilly regions, the gains of which attenuated after the unlocking was initiated. Despite these early gains, the rate of fatalities is significantly higher during the lockdown period in comparison to the plains. The findings remain robust to multiple alternative specifications and methods including one that accounts for confounding possibilities via unobservable and provides consistent estimates of bias adjusted treatment effects. The evidence supports the need for provisioning of public health services and infrastructure upgradation, especially maintenance of adequate stock of life support devices, in high altitude regions. It also underscores the necessity for strengthening and revising the existing Hill Areas Development Programme and integrating important aspects of public health as part of this policy.

Interferon-gamma and perforin-positive T cells in acquired aplastic anemia: implication in therapeutic response.

Acquired aplastic anemia (aAA) is an autoimmune disease, characterized by infiltration of T lymphocytes in the bone marrow with destruction of hematopoietic stem cells by the effector cells. Interferon-gamma (IFN-γ) and perforin are important mediators of cell destruction. In this flow cytometry-based study, we have investigated the percentage of intracellular IFN-γ+ and perforin+ CD5+ T cells in peripheral blood of newly diagnosed aAA patients before and after immunosuppressive therapy (IST). Patients were categorized as per standard disease severity and response to IST. The median percentage of IFN-γ+ and perforin+ CD5+ T cells was higher in untreated patients compared to healthy controls. The percentage of these cells was also increased in untreated severe and very severe aplastic anemia when compared with non-severe aplastic anemia patients. In patients before and after IST the median percentage of T cells producing IFN-γ and perforin was elevated in non-responders as compared to partial plus complete responders. The higher percentage of IFN-γ+ and perforin+ CD5+ T cells may be useful as an early diagnostic marker for aberrant activation of immune system and predict poor response to IST in aAA patients, who will benefit from alternative therapy.

Gαq modulates the energy metabolism of osteoclasts.

Introduction: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs. Methods: The proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function. Results: PMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid. Discussion: Our study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.

Interleukin-9 Facilitates Osteoclastogenesis in Rheumatoid Arthritis.

In rheumatoid arthritis (RA), inflammatory cytokines play a pivotal role in triggering abnormal osteoclastogenesis leading to articular destruction. Recent studies have demonstrated enhanced levels of interleukin-9 (IL-9) in the serum and synovial fluid of patients with RA. In RA, strong correlation has been observed between tissue inflammation and IL-9 expression in synovial tissue. Therefore, we investigated whether IL-9 influences osteoclastogenesis in patients with RA. We conducted the study in active RA patients. For inducing osteoclast differentiation, mononuclear cells were stimulated with soluble receptor activator of NF-kB ligand (sRANKL) and macrophage-colony-stimulating factor (M-CSF) in the presence or absence of recombinant (r) IL-9. IL-9 stimulation significantly enhanced M-CSF/sRANKL-mediated osteoclast formation and function. Transcriptome analysis revealed differential gene expression induced with IL-9 stimulation in the process of osteoclast differentiation. IL-9 mainly modulates the expression of genes, which are involved in the metabolic pathway. Moreover, we observed that IL-9 modulates the expression of matrix metalloproteinases (MMPs), which are critical players in bone degradation. Our results indicate that IL-9 has the potential to influence the structural damage in the RA by promoting osteoclastogenesis and modulating the expression of MMPs. Thus, blocking IL-9 pathways might be an attractive immunotherapeutic target for preventing bone degradation in RA.

Early and Sustained Expansion of Adaptive Natural Killer Cells Following Haploidentical Transplantation and CTLA4Ig-Primed Donor Lymphocyte Infusions Dissociate Graft-versus-Leukemia and Graft-versus-Host Effects.

BACKGROUND: Adaptive or memory natural killer (NK) cells with epigenetic imprints similar to memory T cells have been shown to develop in response to cytomegalovirus (CMV) infection with upregulation of activating receptor NKG2C. These cells have been shown to possess strong anti-tumour efficacy both in-vitro as well as in-vivo. OBJECTIVES: To determine if reconstitution of adaptive NK cells (CD56dimNKG2C+NKG2A-) in patients with advanced leukemia undergoing haploidentical HCT had any impact on disease progression (DP). STUDY DESIGN: The study cohort comprised of 60 patients with advanced acute leukemia, aged 2-65 years, receiving myeloablative PTCy based haploidentical transplantation from CMV seropositive donors, followed by CTLA4Ig-primed donor lymphocyte infusions (DLI). They were evaluated for the kinetics of reconstitution of adaptive NK cells, both phenotypic and functional, at days +30,+60, +90 and at regular intervals, to 3 years of follow-up, in relation to DP. Reconstitution of adaptive NK cells was compared with a retrospective cohort of patients in the same protocol receiving DLI without CTLA4Ig. RESULTS: Non-relapse mortality, acute and chronic GVHD were 5.1%, 10.3% and 14.5%. DP was 17.5% at a median follow-up of 28 months. Adaptive NK cells were significantly higher in patients without DP at days+30, +60 and +90 (p = 0.0001), irrespective of CMV reactivation and remained elevated until 36 months post-HCT. These cells maintained their functional competence as measured by robust interferon-gamma production with higher expressions of KIR, NKG2D and CD57, without any increase in PD1 expression. Grafts from donors with higher adaptive NK cells were associated with a lower risk of DP (p = 0.0001). In multivariate analysis, adaptive NK cell recovery at day +90 had the most favorable impact on DP (HR-0.7). Tregs reconstituted briskly along with the adaptive NK cells and were sustained as well, without compromising the GVL effect. Comparison with a retrospective cohort receiving the same protocol with DLI without CTLA4Ig, showed a superior reconstitution of adaptive NK cells in those receiving CTLA4Ig-DLI (p < 0.0001). CONCLUSION: Our study suggests that myeloablative transplantation from CMV seropositive haploidentical donors augmented with CTLA4Ig-primed DLI might favor early and sustained expansion of functionally competent adaptive NK cells irrespective of CMV reactivation, with a favorable outcome.

Inhibiting OX40 Restores Regulatory T-Cell Function and Suppresses Inflammation in Pulmonary Sarcoidosis.

BACKGROUND: Pulmonary sarcoidosis (PS) is a noncaseating granulomatous disease of unknown origin. Despite conflicting reports, it is considered that the regulatory T (Treg) cells are functionally impaired in PS, but the underlying mechanisms remain unclear. OX40, a pivotal costimulatory molecule, is essential for T-cell functions and memory development, but its impact on Treg cells is ambiguous. RESEARCH QUESTION: Does the OX40 pathway influence the suppressive functions of Treg cells in PS? STUDY DESIGN AND METHODS: Fifty treatment-naïve patients with PS and 30 healthy control participants were recruited for this study. Polychromatic flow cytometry-based immunologic assays were performed to enumerate effector T helper (Th) cells and Treg cells along with their functions. Using real-time polymerase chain reaction analysis, small interfering RNA, and pharmacologic inhibitors, the impact of OX40 on Treg cell function was investigated. RESULTS: We observed enrichment of Th-9 cells perhaps for the first time along with Th-1, Th-17, and Treg cells in patients' BAL fluid (BALF) compared with peripheral blood. However, Treg cells were observed to be functionally defective at the pathological site. We observed higher expression of OX40 on both T effector (CD4+Foxp3-) and Treg (CD4+Foxp3+) cells obtained from the BALF of patients with PS. However, OX40 exerted contrasting impact on these T-cell subsets, enhancing effector T-cell functions (interferon γ, tumor necrosis factor α) while inhibiting Treg cell function (IL-10, transforming growth factor ß). OX40 silencing or blocking on Treg cells resulted in restoration of their impaired functions. INTERPRETATION: We propose that inhibiting the OX40 pathway may constitute a therapeutic strategy for controlling inflammatory T cells by restoring Treg cell functions in patients with PS.

Successful reconstitution of leukocyte adhesion defect after umbilical cord blood stem cell transplant.

Leukocyte adhesion deficiencies (LADs) are a type of primary immunodeficiencies characterized by delayed detachment of the umbilical cord, impaired wound healing, leukocytosis, and recurrent infections. The disease is caused by genetic defects affecting different steps in the process of leukocyte adhesion cascade such as rolling, integrin activation, and adhesion of leukocytes, resulting in the impairment of leukocyte trafficking. Till date, three types of LAD have been documented: type I, II and III. Type I LAD is caused by congenital defect in the ß2 integrin receptor complex CD11/CD18 on the cell surface of leukocytes, which results in impaired leukocytes connection to endothelial cells and migration. Type II LAD is caused by defect in the fucose metabolism resulting in the absence of fucosylated selectin ligands on neutrophils and impaired rolling phase of the leukocyte adhesion cascade. Type III LAD is caused by mutations in the kindlin-3 gene resulting in defective integrin activation. In this article, we present a review of literature for type I LAD, and successful treatment of patient using umbilical cord blood stem cell transplantation.

Post Transplant Lymphoproliferative Disorder.

Posttransplant lymphoproliferative disorder is an extremely fatal complication arising in transplant recipients as a side effect of immunosuppression. PTLDs are seen after both solid organ and hematopoietic stem cell transplants though the incidence is much higher in the former. Primary Epstein-Barr virus (EBV) infection or reactivation due to a state of immune dysregulation along with intensity of immunosuppression used are of paramount importance in pathogenesis of PTLD. EBV associated PTLDs occur early in the post transplant period whereas late onset lymphomas are usually EBV negative. The uncontrolled B cell proliferation can create a spectrum of histological patterns from nondestructive lesions to destructive polymorphic or more aggressive monomorphic PTLDs. Early detection of seropositivity by serial monitoring in the recipient can prevent PTLD development by starting pre-emptive therapy. The mainstay treatment in established cases remains reduction of immunosuppression. Chemotherapeutic and immunomodulatory agents are added sequentially based on the type of PTLD and based on its response to initial therapy. Despite various treatment options available, the morbidity remains high and achieving state of disease remission without causing graft rejection can be quite challenging. Hence, a better understanding in pathobiology of EBV+ versus EBV- PTLDS may help prevent lymphomagenesis in transplant recipients.

Ras isoforms: signaling specificities in CD40 pathway.

BACKGROUND: Ras are small cellular GTPases which regulate diverse cellular processes. It has three isoforms: H-Ras, K-Ras, and N-Ras. Owing to the N-terminus (1-165 residues) sequence homology these isoforms were thought to be functionally redundant. However, only K-Ras-deficient mice but not H-Ras- and N-Ras-deficient mice show embryonic lethality. Similarly, mutations in a given Ras isoform are associated with a particular type of cancer. Moreover, we have previously reported that Ras isoforms perform unique functions in Leishmania major infection. Thus, Ras isoforms are implicated to have signaling and functional specificity but the mechanism remains to be elucidated. RESULT: Using CD40 as a model receptor, we showed that depending on the strength of signaling, specific Ras isoforms are activated. Weak CD40 signal activates N-Ras, whereas strong signal activates H-Ras and K-Ras. Additionally, we showed that suppression of N-Ras expression reduced CD40-induced extracellular signal-regulated kinase-1/2 (ERK-1/2) activation and Interleukin (IL)-10 production; whereas suppression of H-Ras or K-Ras reduced CD40-induced p38 mitogen-activated protein kinase (p38MAPK) activation and IL-12 production. Furthermore, we showed that Ras isoforms have activator (GEF) specificity as weak CD40 signal-activated N-Ras requires Sos-1/2 whereas strong CD40 signal-activated H-Ras/K-Ras requires Ras-GRP as the guanine-nucleotide exchange factor (GEF) inducing ERK-1/2- or p38MAPK-mediated IL-10 or IL-12 productions, respectively, in macrophages. Silencing of syk reduced CD40-induced N-Ras activation but silencing of lyn inhibited H-Ras and K-Ras activation. In CD40 signaling, Ras isoforms also showed effector specificity; while H-Ras and K-Ras showed specificity for phosphatidyl inositol-3 kinase activation at high dose of CD40 stimulation, N-Ras primarily associated with Raf-1 at low dose of CD40 stimulation. Moreover, fractal analysis showed that functional site surface roughness for H-Ras (SurfaceFD = 2.39) and K-Ras (SurfaceFD = 2.39) are similar but significantly different from N-Ras (SurfaceFD = 2.25). CONCLUSION: The activator and effector specificities of Ras isoforms in CD40 signaling indicated their differential involvement in CD40 pathway and in maintaining the reciprocity. Our observations reveal Ras-regulated signaling outcome and its potential for developing Ras isoform-targeted immunotherapy and prophylaxis.

Primary Immunodeficiency Disorders Among North Indian Children.

OBJECTIVES: To report the distribution pattern of various categories of primary immunodeficiency disorders (PIDs) in children from North India, frequency of warning signs and critical parameters for evaluation. METHODS: In this retrospective study, 528 children below 18 y of age after clinical assessment and presentation suggestive of PID were further screened by immunophenotyping for immune cell markers by flow cytometry. RESULTS: A total of 120 (23%) children were diagnosed with PID with median age at diagnosis being 2.5 y in males and 3.5 y in females and an average delay in diagnosis from onset of symptoms being approximately 5 y. Chronic lower respiratory tract infections, gastrointestinal symptoms like persistent diarrhea and failure to thrive were amongst the most common warning signs in these patients. PIDs were classified according to the International Union of Immunological Societies' (IUIS) criteria. The diagnosis of index study subjects included combined humoral and cellular immunodeficiency (29%), phagocytic defects (29%), followed by predominantly antibody deficiency (18%), innate immunity and dysregulation (17%) and other well-defined syndromes (7%). A family history of PID (23%), consanguineous marriage (8%) and previous sibling death (23%) were observed as major clinical predictors/clues for underlying PID. All children received prophylactic antibiotics and/or antifungals in addition to specific therapy for underlying immune deficiency. CONCLUSIONS: The field of PIDs in India remains largely unexplored and we are faced with various challenges in the diagnosis of PIDs due to lack of awareness as well as absence of equipped immunological laboratory support. The authors propose a methodical step-wise laboratory diagnostic approach that can facilitate early diagnosis and timely intervention of these mis/underdiagnosed disorders.

An Update on Interleukin-9: From Its Cellular Source and Signal Transduction to Its Role in Immunopathogenesis.

Interleukin-9 (IL-9) is a pleiotropic cytokine and was primarily studied in the context of T helper 2 (TH2)-associated immuno-pathological conditions such as asthma and parasitic infections. There was a paradigm shift in the biology of IL-9 after the recent discovery of TH9 cells, a new subtype of TH cells which secrete IL-9 in copious amounts. This has resulted in renewed interest in this cytokine, which was neglected since discovery because it was considered it to be just another TH2 cytokine. Recent studies have shown that it has multiple cellular sources and is critically involved in the immune-pathogenesis of inflammatory diseases and in guarding immune tolerance. In this review, we will discuss its discovery, gene organization, cellular sources, and signaling pathways. Especially, we will give an update on the recent development regarding its relevance in the immune pathogenesis of human diseases.

Inhibiting HLA-G restores IFN-γ and TNF-α producing T cell in pleural Tuberculosis.

Human Leukocyte Antigen-G (HLA-G), a non-classical, class Ib molecule, has been shown to mediate immunoregulatory functions by inducing apoptosis, inhibits cytotoxicity and differentiation by modulating cytokine secretion. Due to its immune-suppressive function, it facilitates tolerance in feto-maternal interface and transplantation. In contrary, it favours immune evasion of microbes and tumors by inhibiting immune and inflammatory responses. In Tuberculosis (TB), we previously reported differential expression of HLA-G and its receptor Ig-like transcript -2 (ILT-2) in disseminated vs. localized Tuberculosis. The present study explores the impact of HLA-G inhibition on the function of T cells and monocytes, in TB Pleural Effusion (PE), a localized form of TB. Blocking of HLA-G resulted in significant increase in IFN-γ and TNF-α production by CD3+ T cells. Additionally, we observed that HLA-G influences the apoptosis and cytotoxic effect of T cells from TB- PE patients. Next, we checked the impact of interaction between HLA-G and ILT-4 receptor in monocytes derived from TB-PE patients upon blocking and observed significant increase in IFN-γ production. The present study reveals for the first time HLA-G mediated suppression of Th1 cytokines, especially, IFN-γ and TNF-α in TB-PE patients.

Influence of Pasteurella multocida Toxin on the differentiation of dendritic cells into osteoclasts.

Dendritic cells (DC) are antigen-presenting cells that connect the innate and adaptive immune system to ensure an efficient immune response during the course of an infection. Recently, DC came into the spotlight as a potential source of osteoclast progenitors, especially under (auto)inflammatory conditions. The virulence factor Pasteurella multocida Toxin (PMT) causes atrophic rhinitis in pigs, a disease characterised by a severe reduction of nasal bone. Our group and others have shown the potential of PMT in mediating differentiation of monocytes/macrophages into bone-resorbing osteoclasts. However, whether DC are target cells for PMT-induced osteoclast differentiation, is currently unknown. Using different murine DC model systems, we investigated the ability of PMT to induce osteoclast formation in DC. Similar to our previous observations in macrophages, PMT was endocytosed by DC and triggered intracellular deamidation of residue Q209 of the Gq alpha subunit. Still, PMT failed to induce prolonged secretion of osteoclastogenic cytokines and osteoclast formation; instead PMT-treated DC secreted interleukin-12 (IL-12), an inhibitor of osteoclastogenesis. In this study, we show that in comparison to bone marrow-derived macrophages, PMT induces maturation of DC through increased expression of the activation markers CD80 and CD86. As maturation of DC prevents their transdifferentiation into osteoclasts, we hypothesize that PMT, a potent osteoclastogenic toxin, fails to trigger osteoclastogenesis in DC due to its effect on DC maturation and IL-12 production.

Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis.

Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.