VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection.

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with ΔvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with ΔvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of ΔvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the ΔvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.

Kennedy Epitope (KE)-dependent Retrograde Transport of Efficiently Cleaved HIV-1 Envelopes (Envs) and its Effect on Env Cell Surface Expression and Viral Particle Formation.

Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 - 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 - 2.J41 and JRCSF Envs increases [as does in the presence of Rab7a DN and Rab7b DN (DN: dominant negative)] but particle formation decreases for 4 - 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes.

Heavy and less common metals in rainwater and the kinetics of the oxidation of hydrogen sulfide by oxygen in rainwater medium.

As a sequel to our recent study on the oxidation by O2, i. e., autoxidation of dissolved hydrogen sulfide, H2S, in aqueous medium, we present here the results of a similar study in rain water medium. The rainwater sampling was done sequentially for four years (2016-2019). In all 67 samples of rainwater were collected on event basis during monsoon period (June-October) in and around the Campus of University of Rajasthan, Jaipur (26°50̛ N-75°52̛ E), which is situated in a semi-arid region adjacent to the Thar Desert in the North-Western part of the India having an annual average rainfall of 54 cm. ICP-MS technique was used in the analysis of rainwater and more than 30 metals were detected, which included transition metals, rare earths, less common metals. Beryllium, vanadium, silver, selenium, manganese, cerium, gallium, yttrium, barium, cesium, copper, rubidium, arsenic, lanthanum, cadmium, lead, uranium and bismuth were common to all rain water samples.H2S oxidation by dissolved oxygen was studied in rainwater as well as in laboratory water media for comparison. The kinetics rate law was: -d[O2]/dt = k1K1[H+][S][O2]t/( [H+]2 + K1[H+] + K1K2), where K1 is first dissociation constant of H2S and k1 is the rate constant for the dominant reaction step:HS- + O2 → products.

Formic acid, an organic acid food preservative, induces viable-but-non-culturable state, and triggers new Antimicrobial Resistance traits in Acinetobacter baumannii and Klebsiella pneumoniae.

Numerous human pathogens, especially Gram-negative bacteria, are able to enter the viable-but-non-culturable (VBNC) state when they are exposed to environmental stressors and pose the risk of being resuscitated and causing infection after the removal of the trigger. Widely used food preservatives like weak organic acids are potential VBNC inducers in food processing and packaging facilities but have only been reported for food-borne pathogens. In the present study, it is demonstrated for the first time that one such agent, formic acid (FA), can induce a VBNC state at food processing, storage, and distribution temperatures (4, 25, and 37°C) with a varied time of treatment (days 4-10) in pathogenic Gram-negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. The use of hospital-associated pathogens is critical based on the earlier reports that demonstrated the presence of these bacteria in hospital kitchens and commonly consumed foods. VBNC induction was validated by multiple parameters, e.g., non-culturability, metabolic activity as energy production, respiratory markers, and membrane integrity. Furthermore, it was demonstrated that the removal of FA was able to resuscitate VBNC with an increased expression of multiple virulence and Antimicrobial Resistance (AMR) genes in both pathogens. Since food additives/preservatives are significantly used in most food manufacturing facilities supplying to hospitals, contamination of these packaged foods with pathogenic bacteria and the consequence of exposure to food additives emerge as pertinent issues for infection control, and control of antimicrobial resistance in the hospital setting.

Study of Yang's Onion Flap Technique for Release of Scarred Eponychium and Nail Fold Reconstruction in Burn Patients.

Background Abnormalities of fingernail growth and appearance are among the most common deformities encountered after burn injury to the hand. Various techniques used for resurfacing defects include incision of the scarred eponychium and advancement of the distal segment, flap reconstruction-distally, and proximally based transposition/advancement flaps, composite graft techniques, microvascular transfer. In the present study, we used an onion flap to release scarred eponychium and nail fold reconstruction in a single stage without using soft tissue from another area. Materials and methods Forty-four burnt fingers were operated using Yang's onion flap technique. Patients were assessed for flap necrosis, hematoma and infection in the early postoperative period and for donor site scar, nail appearance, and symptomatic relief in a follow-up for at least 4 months. Results The flap was successfully performed on all fingers. Only two fingers had flap necrosis. There was no incidence of hematoma or infection. The donor site scar and nail plate appearance improved and was acceptable to most patients after surgery. There was also significant relief in daily activities in 19 out of 28 symptomatic patients. Conclusion Yang's flap to correct nail deformities in burn patients is feasible in Indian scenario. It is associated with a low complication rate and improved nail appearance. There is also significant symptomatic relief in performing daily activities after surgery.

Proinflammatory Innate Cytokines and Distinct Metabolomic Signatures Shape the T Cell Response in Active COVID-19.

The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young, mildly symptomatic, active COVID-19 patients infected during the first wave in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the delta variant, compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection, in response to TLR 3/7/8-mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-18, IL-1ß, and IL-23, and the abundance of plasma metabolites involved in arginine biosynthesis were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce a long-lasting anti-SARS-CoV-2-specific T cell response.

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant.

Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

Golden Syrian hamster as a model to study cardiovascular complications associated with SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the Golden Syrian hamster causes lung pathology that resembles human coronavirus disease (COVID-19). However, extrapulmonary pathologies associated with SARS-CoV-2 infection and post-COVID sequelae remain to be understood. Here, we show, using a hamster model, that the early phase of SARS-CoV-2 infection leads to an acute inflammatory response and lung pathologies, while the late phase of infection causes cardiovascular complications (CVCs) characterized by ventricular wall thickening associated with increased ventricular mass/body mass ratio and interstitial coronary fibrosis. Molecular profiling further substantiated our findings of CVC as SARS-CoV-2-infected hamsters showed elevated levels of serum cardiac troponin I, cholesterol, low-density lipoprotein, and long-chain fatty acid triglycerides. Serum metabolomics profiling of SARS-CoV-2-infected hamsters identified N-acetylneuraminate, a functional metabolite found to be associated with CVC, as a metabolic marker was found to be common between SARS-CoV-2-infected hamsters and COVID-19 patients. Together, we propose hamsters as a suitable animal model to study post-COVID sequelae associated with CVC, which could be extended to therapeutic interventions.

Effectiveness of ChAdOx1 nCoV-19 vaccine against SARS-CoV-2 infection during the delta (B.1.617.2) variant surge in India: a test-negative, case-control study and a mechanistic study of post-vaccination immune responses.

BACKGROUND: SARS-CoV-2 variants of concern (VOCs) have threatened COVID-19 vaccine effectiveness. We aimed to assess the effectiveness of the ChAdOx1 nCoV-19 vaccine, predominantly against the delta (B.1.617.2) variant, in addition to the cellular immune response to vaccination. METHODS: We did a test-negative, case-control study at two medical research centres in Faridabad, India. All individuals who had a positive RT-PCR test for SARS-CoV-2 infection between April 1, 2021, and May 31, 2021, were included as cases and individuals who had a negative RT-PCR test were included as controls after matching with cases on calendar week of RT-PCR test. The primary outcome was effectiveness of complete vaccination with the ChAdOx1 nCoV-19 vaccine against laboratory-confirmed SARS-CoV-2 infection. The secondary outcomes were effectiveness of a single dose against SARS-CoV-2 infection and effectiveness of a single dose and complete vaccination against moderate-to-severe disease among infected individuals. Additionally, we tested in-vitro live-virus neutralisation and T-cell immune responses to the spike protein of the wild-type SARS-CoV-2 and VOCs among healthy (anti-nucleocapsid antibody negative) recipients of the ChAdOx1 nCoV-19 vaccine. FINDINGS: Of 2379 cases of confirmed SARS-CoV-2 infection, 85 (3·6%) were fully vaccinated compared with 168 (8·5%) of 1981 controls (adjusted OR [aOR] 0·37 [95% CI 0·28-0·48]), giving a vaccine effectiveness against SARS-CoV-2 infection of 63·1% (95% CI 51·5-72·1). 157 (6·4%) of 2451 of cases and 181 (9·1%) of 1994) controls had received a single dose of the ChAdOx1 nCoV-19 vaccine (aOR 0·54 [95% CI 0·42-0·68]), thus vaccine effectiveness of a single dose against SARS-CoV-2 infection was 46·2% (95% CI 31·6-57·7). One of 84 cases with moderate-to-severe COVID-19 was fully vaccinated compared with 84 of 2295 cases with mild COVID-19 (aOR 0·19 [95% CI 0·01-0·90]), giving a vaccine effectiveness of complete vaccination against moderate-to-severe disease of 81·5% (95% CI 9·9-99·0). The effectiveness of a single dose against moderate-to-severe disease was 79·2% (95% CI 46·1-94·0); four of 87 individuals with moderate-to-severe COVID-19 had received a single dose compared with 153 of 2364 participants with mild disease (aOR 0·20 [95% CI 0·06-0·54]). Among 49 healthy, fully vaccinated individuals, neutralising antibody responses were lower against the alpha (B.1.1.7; geometric mean titre 244·7 [95% CI 151·8-394·4]), beta (B.1.351; 97·6 [61·2-155·8]), kappa (B.1.617.1; 112·8 [72·7-175·0]), and delta (88·4 [61·2-127·8]) variants than against wild-type SARS-CoV-2 (599·4 [376·9-953·2]). However, the antigen-specific CD4 and CD8 T-cell responses were conserved against both the delta variant and wild-type SARS-CoV-2. INTERPRETATION: The ChAdOx1 nCoV-19 vaccine remained effective against moderate-to-severe COVID-19, even during a surge that was dominated by the highly transmissible delta variant of SARS-CoV-2. Spike-specific T-cell responses were maintained against the delta variant. Such cellular immune protection might compensate for waning humoral immunity. FUNDING: Department of Biotechnology India, Council of Scientific and Industrial Research India, and Fondation Botnar.

The SARS CoV-2 spike directed non-neutralizing polyclonal antibodies cross-react with Human immunodeficiency virus (HIV-1) gp41.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

Activation of epigenetic regulator KDM6B by Salmonella Typhimurium enables chronic infections.

Non-typhoidal Salmonella (NTS) infections result in self limiting gastroenteritis except in rare cases wherein manifestations of chronic infections can occur. Strategies employed by Salmonella to thrive in hostile environments of host during chronic infections are complex and multifaceted. In chronic state, a coordinated action of bacterial effectors allows reprogramming of macrophages to M2 subtype and thereby creating a permissible replicative niche. The mechanistic details of these processes are not fully known. In the current study we identified, histone H3-lysine 27 trimethylation (H3K27me3)-specific demethylase, KDM6B to be upregulated in both cell culture and in murine model of Salmonella infection. KDM6B recruitment upon infection exhibited an associated loss of overall H3K27me3 in host cells and was Salmonella SPI1 effectors coordinated. ChIP-qRT-PCR array analysis revealed several new gene promoter targets of KDM6B demethylase activity including PPARδ, a crucial regulator of fatty acid oxidation pathway and Salmonella-persistent infections. Furthermore, pharmacological inhibition of KDM6B demethylase activity with GSKJ4 in chronic Salmonella infection mice model led to a significant reduction in pathogen load and M2 macrophage polarization in peripheral lymphoid organs. The following work thus reveals Salmonella effector-mediated epigenetic reprogramming of macrophages responsible for its long-term survival and chronic carriage.

High-salt diet mediates interplay between NK cells and gut microbiota to induce potent tumor immunity.

High-salt diet (HSD) modulates effector and regulatory T cell functions and promotes tissue inflammation in autoimmune diseases. However, effects of HSD and its association with gut microbiota in tumor immunity remain undefined. Here, we report that HSD induces natural killer (NK) cell­mediated tumor immunity by inhibiting PD-1 expression while enhancing IFNγ and serum hippurate. Salt enhanced tumor immunity when combined with a suboptimal dose of anti-PD1 antibody. While HSD-induced tumor immunity was blunted upon gut microbiota depletion, fecal microbiota transplantation (FMT) from HSD mice restored the tumor immunity associated with NK cell functions. HSD increased the abundance of Bifidobacterium and caused increased gut permeability leading to intratumor localization of Bifidobacterium, which enhanced NK cell functions and tumor regression. Intratumoral injections of Bifidobacterium activated NK cells, which inhibited tumor growth. These results indicate that HSD modulates gut microbiome that induces NK cell­dependent tumor immunity with a potential translational perspective.

Japanese Encephalitis Virus Infected Human Monocyte-Derived Dendritic Cells Activate a Transcriptional Network Leading to an Antiviral Inflammatory Response.

A comprehensive understanding of the human immune response to virus infection is imperative for developing effective therapies, antivirals, and vaccines. Dendritic cells (DCs) are among the first cells to encounter the virus and are also key antigen-presenting cells that link the innate and adaptive immune system. In this study, we focus on the human immune response to the mosquito-borne Japanese encephalitis virus (JEV), which is the leading cause of virus-induced encephalitis in south-east Asia and has the potential to become a global pathogen. We describe the gene regulatory circuit of JEV infection in human monocyte-derived DCs (moDCs) along with its functional validation. We observe that JEV can productively infect human moDCs leading to robust transcriptional activation of the interferon and NF-κB-mediated antiviral and inflammatory pathways. This is accompanied with DC maturation and release of pro-inflammatory cytokines and chemokines TNFα, IL-6, IL-8, IL-12, MCP-1. and RANTES. JEV-infected moDCs activated T-regulatory cells (Tregs) in allogenic mixed lymphocyte reactions (MLR) as seen by upregulated FOXP3 mRNA expression, suggestive of a host response to reduce virus-induced immunopathology. The virus also downregulated transcripts involved in Peroxisome Proliferator Activated Receptor (PPAR) signalling and fatty acid metabolism pathways suggesting that changes in cellular metabolism play a crucial role in driving the DC maturation and antiviral responses. Collectively, our data describe and corroborate the human DC transcriptional network that is engaged upon JEV sensing.

Gefitinib Results in Robust Host-Directed Immunity Against Salmonella Infection Through Proteo-Metabolomic Reprogramming.

The global rise of antibiotic-resistant strains of Salmonella has necessitated the development of alternative therapeutic strategies. Recent studies have shown that targeting host factors may provide an alternative approach for the treatment of intracellular pathogens. Host-directed therapy (HDT) modulates host cellular factors that are essential to support the replication of the intracellular pathogens. In the current study, we identified Gefitinib as a potential host directed therapeutic drug against Salmonella. Further, using the proteome analysis of Salmonella-infected macrophages, we identified EGFR, a host factor, promoting intracellular survival of Salmonella via mTOR-HIF-1α axis. Blocking of EGFR, mTOR or HIF-1α inhibits the intracellular survival of Salmonella within the macrophages and in mice. Global proteo-metabolomics profiling indicated the upregulation of host factors predominantly associated with ATP turn over, glycolysis, urea cycle, which ultimately promote the activation of EGFR-HIF1α signaling upon infection. Importantly, inhibition of EGFR and HIF1α restored both proteomics and metabolomics changes caused by Salmonella infection. Taken together, this study identifies Gefitinib as a host directed drug that holds potential translational values against Salmonella infection and might be useful for the treatment of other intracellular infections.

Replication of Dengue Virus in K562-Megakaryocytes Induces Suppression in the Accumulation of Reactive Oxygen Species.

Dengue virus can infect human megakaryocytes leading to decreased platelet biogenesis. In this article, we report a study of Dengue replication in human K562 cells undergoing PMA-induced differentiation into megakaryocytes. PMA-induced differentiation in these cells recapitulates steps of megakaryopoiesis including gene activation, expression of CD41/61 and CD61 platelet surface markers and accumulation of intracellular reactive oxygen species (ROS). Our results show differentiating megakaryocyte cells to support higher viral replication without any apparent increase in virus entry. Further, Dengue replication suppresses the accumulation of ROS in differentiating cells, probably by only augmenting the activity of the transcription factor NFE2L2 without influencing the expression of the coding gene. Interestingly pharmacological modulation of NFE2L2 activity showed a simultaneous but opposite effect on intracellular ROS and virus replication suggesting the former to have an inhibitory effect on the later. Also cells that differentiated while supporting intracellular virus replication showed reduced level of surface markers compared to uninfected differentiated cells.

Absence of Nonclassical Monocytes in Hemolytic Patients: Free Hb and NO-Mediated Mechanism.

In a recent work, we have described the kinetics among the monocyte subsets in the peripheral blood of hemolytic patients including paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD). After engulfing Hb-activated platelets, classical monocytes (CD14+CD16-) significantly transformed into highly inflammatory (CD14+CD16hi) subsets in vitro. An estimated 40% of total circulating monocytes in PNH and 70% in SCD patients existed as CD14+CD16hi subsets. In this study, we show that the nonclassical (CD14dimCD16+) monocyte subsets are nearly absent in patients with PNH or SCD, compared to 10-12% cells in healthy individuals. In mechanism, we have described the unique role of both free Hb and nitric oxide (NO) in reducing number of nonclassical subsets more than classical monocytes. After engulfing Hb-activated platelets, the monocytes including nonclassical subsets acquired rapid cell death within 12 h in vitro. Further, the treatment to monocytes either with the secretome of Hb-activated platelets containing NO and free Hb or purified free Hb along with GSNO (a physiological NO donor) enhanced rapid cell death. Besides, our data from both PNH and SCD patients exhibited a direct correlation between intracellular NO and cell death marker 7AAD in monocytes from the peripheral blood. Our data together suggest that due to the immune surveillance nature, the nonclassical or patrolling monocytes are encountered frequently by Hb-activated platelets, free Hb, and NO in the circulation of hemolytic patients and are predisposed to die rapidly.

Differences in multiple immune parameters between Indian and U.S. infants.

To compare immune phenotypes across two geographic and ethnic communities, we examined umbilical cord blood by flow cytometry and Luminex in parallel cohorts of 53 newborns from New Delhi, India, and 46 newborns from Stanford, California. We found that frequencies of a B cell subset suggested to be B-1-like, and serum IgM concentration were both significantly higher in the Stanford cohort, independent of differences in maternal age. While serum IgA levels were also significantly higher in the Stanford cohort, IgG1, IgG2, and IgG4 were significantly higher in the New Delhi samples. We found that neutrophils, plasmacytoid dendritic cells, CD8+ T cells, and total T cells were higher in the U.S. cohort, while dendritic cells, patrolling monocytes (CD14dimCD16+), natural killer cells, CD4+ T cells, and naïve B cells were higher in the India cohort. Within the India cohort, we also identified cell types whose frequency was positively or negatively predictive of occurrence of infection(s) in the first six months of life. Monocytes, total T cells, and memory CD4+ T cells were most prominent in having an inverse relationship with infection. We suggest that these data provide impetus for follow-up studies linking phenotypic differences to environmental versus genetic factors, and to infection outcomes.

Engulfment of Hb-activated platelets differentiates monocytes into pro-inflammatory macrophages in PNH patients.

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)-activated platelets, classical monocytes differentiated into pro-inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb-activated platelets, monocytes differentiate into M1-macrophages under M1-polarization stimulus (GM-CSF, IFN-γ + LPS). When grown under M2-polarization stimulus (M-CSF, IL-4 + IL13), the cells exhibited an M1-like phenotype, secreted elevated levels of pro-inflammatory cytokines including TNF-α and IL-1ß, and displayed loss of the secretion of cytokine such as IL-10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho-STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte-platelet aggregates in circulation and accumulation of CD80high macrophages in thioglycollate-induced inflamed peritoneum. The spleen of the mice was also populated by CD80high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb-activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.

Transcription factor Foxo1 is essential for IL-9 induction in T helper cells.

Interleukin 9 (IL-9)-producing helper T (Th9) cells have a crucial function in allergic inflammation, autoimmunity, immunity to extracellular pathogens and anti-tumor immune responses. In addition to Th9, Th2, Th17 and Foxp3+ regulatory T (Treg) cells produce IL-9. A transcription factor that is critical for IL-9 induction in Th2, Th9 and Th17 cells has not been identified. Here we show that the forkhead family transcription factor Foxo1 is required for IL-9 induction in Th9 and Th17 cells. We further show that inhibition of AKT enhances IL-9 induction in Th9 cells while it reciprocally regulates IL-9 and IL-17 in Th17 cells via Foxo1. Mechanistically, Foxo1 binds and transactivates IL-9 and IRF4 promoters in Th9, Th17 and iTreg cells. Furthermore, loss of Foxo1 attenuates IL-9 in mouse and human Th9 and Th17 cells, and ameliorates allergic inflammation in asthma. Our findings thus identify that Foxo1 is essential for IL-9 induction in Th9 and Th17 cells.The transcription factor Foxo1 can control regulatory T cell and Th1 function. Here the authors show that Foxo1 is also critical for IL-9 production by Th9 cells and other IL-9-producing cells.