Inflammasome activation in patients with Kaposi sarcoma herpesvirus (KSHV)-associated disorders.

Kaposi sarcoma herpesvirus (KSHV)-associated disorders include Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated multicentric Castleman disease (MCD) and KSHV-inflammatory cytokine syndrome (KICS). PEL, MCD, and KICS are associated with elevated circulating inflammatory cytokines. However, activation of the inflammasome, which generates IL-1 and IL-18 via active caspase-1/4/5, has not been evaluated in patients with KAD. Here we report that patients with HIV and one or more KAD present with higher plasma levels of IL-18 and increased caspase-1/4/5 activity in circulating monocytes as compared to HIV-negative healthy volunteers (HV) or people with HIV without KAD (PWH). Within KAD subtypes, KICS and MCD shared enhanced caspase-1/4/5 activity and IL-18 production when compared to HV and PWH, while patients with PEL showed remarkably high levels of inflammasome complex formation (known as apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC)-speck). Moreover, caspase-1/4/5 activity and IL-18 plasma levels correlated with KSHV viral load, indicating KSHV-driven inflammasome activation in KAD. Accordingly, factors released by KSHV-latently infected cells triggered inflammasome activation and cytokine production in bystander monocytes, in vitro. Finally, both supervised and unsupervised analyses with inflammasome measurements and other inflammatory biomarkers demonstrate a unique inflammatory profile in patients with PEL, MCD, and KICS as compared to KS. Our data indicate that detrimental inflammation in patients with KAD is at least partially driven by KSHV-induced inflammasome activation in monocytes, thus offering novel approaches to diagnose and treat these complex disorders.

Clonal hematopoiesis in people with advanced HIV and associated inflammatory syndromes.

People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts; however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4+ and CD8+ T cells, nadir CD4+ count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197 PWH with median age of 42 years, using a 56-gene panel. Seventy-nine percent had a CD4+ nadir below 200 cells/µL, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8+ T cells and nadir CD4+ counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of patients 35 years of age and older. Inflammatory biomarkers were higher in CH carriers compared with noncarriers, supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4+ and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.

Pyroptosis Markers in Human Primary Specimens: Quantification of Intracellular ASC Specks by Imaging Flow Cytometry and Extracellular Oxidized Mitochondrial by ELISA.

Pyroptosis is an immunological response to infection and cellular stresses initiated by inflammasome oligomerization resulting in the release of pro-inflammatory factors including cytokines and other immune stimuli into the extracellular matrix. In order to understand the role of inflammasome activation and subsequent pyroptosis in human infection and disease pathogenesis and to explore markers of these signaling events as potential disease or response biomarkers, we must utilize quantitative, reliable, and reproducible assays to readily investigate these pathways in primary specimens. Here, we describe two methods using imaging flow cytometry for evaluation of inflammasome ASC specks in homogeneous peripheral blood monocytes and in bulk, heterogeneous peripheral blood mononuclear cells. Both methods can be applied to assess speck formation as a biomarker for inflammasome activation in primary specimens. Additionally, we describe the methods for quantification of extracellular oxidized mitochondrial DNA from primary plasma samples, serving as a proxy for pyroptosis. Collectively, these assays may be utilized to determine pyroptotic influences on viral infection and disease development or as diagnostic aids and response biomarkers.

Polyfunctional Antigen Specific CD4+ T cell Responses in Patients With Human Immunodeficiency Virus/AIDS and Histoplasmosis Immune Reconstitution Inflammatory Syndrome.

In the combination antiretroviral era, there are limited data regarding the pathogenesis of histoplasmosis immune reconstitution inflammatory syndrome (IRIS) in people with human immunodeficiency virus (HIV). We immunologically characterized 10 cases of histoplasmosis, 4 of whom developed histoplasmosis IRIS. CD4+ T cells in histoplasmosis IRIS demonstrated a significant polyfunctional cytokine response to histoplasma antigen.

Lysosomal cathepsins act in concert with Gasdermin-D during NAIP/NLRC4-dependent IL-1ß secretion.

The NAIP/NLRC4 inflammasome is classically associated with the detection of bacterial invasion to the cytosol. However, recent studies have demonstrated that NAIP/NLRC4 is also activated in non-bacterial infections, and in sterile inflammation. Moreover, in addition to the well-established model for the detection of bacterial proteins by NAIP proteins, the participation of other cytosolic pathways in the regulation of NAIP/NLRC4-mediated responses has been reported in distinct contexts. Using pharmacological inhibition and genetic deletion, we demonstrate here that cathepsins, well known for their involvement in NLRP3 activation, also regulate NAIP/NLRC4 responses to cytosolic flagellin in murine and human macrophages. In contrast to that observed for NLRP3 agonists, cathepsins inhibition did not reduce ASC speck formation or caspase-1 maturation in response to flagellin, ruling out their participation in the effector phase of NAIP/NLRC4 activation. Moreover, cathepsins had no impact on NF-κB-mediated priming of pro-IL-1ß, thus suggesting these proteases act downstream of the NAIP/NLRC4 inflammasome activation. IL-1ß levels secreted in response to flagellin were reduced in the absence of either cathepsins or Gasdermin-D (GSDMD), a molecule involved in the induction of pyroptosis and cytokines release. Notably, IL-1ß secretion was abrogated in the absence of both GSDMD and cathepsins, demonstrating their non-redundant roles for the optimal IL-1ß release in response to cytosolic flagellin. Given the central role of NAIP/NLRC4 inflammasomes in controlling infection and, also, induction of inflammatory pathologies, many efforts have been made to uncover novel molecules involved in their regulation. Thus, our findings bring together a relevant contribution by describing the role of cathepsins as players in the NAIP/NLRC4-mediated responses.

Macrophage Polarization in the Perivascular Fat Was Associated With Coronary Atherosclerosis.

Background Inflammation of the perivascular adipose tissue (PvAT) may be related to atherosclerosis; however, the association of polarized macrophages in the pericoronary PvAT with measurements of atherosclerosis components in humans has not been fully investigated. Methods and Results Coronary arteries were dissected with surrounding PvAT. We evaluated the percentage of arterial obstruction, intima-media thickness, fibrous cap thickness, plaque components, and the number of vasa vasorum. The number of proinflammatory (M1) and anti-inflammatory (M2) macrophages in the periplaque and control PvAT were evaluated using immunohistochemistry. Regression models adjusted for sociodemographic and clinical variables were used. In 319 segments from 82 individuals, we found a correlation of the M1/M2 macrophage density ratio with an increase in arterial obstruction (P=0.02) and lipid content (P=0.01), and a decrease in smooth muscle cells (P=0.02). M1 and the ratio of M1/M2 macrophages were associated with an increased risk of thrombosis (P=0.03). In plaques with thrombosis, M1 macrophages were correlated with a decrease in fibrous cap thickness (P=0.006), an increase in lipid content (P=0.008), and the number of vasa vasorum in the adventitia layer (P=0.001). M2 macrophages were correlated with increased arterial obstruction (P=0.01), calcification (P=0.02), necrosis (P=0.03) only in plaques without thrombosis, and decrease of the number of vasa vasorum in plaques with thrombosis (P=0.003). Conclusions M1 macrophages in the periplaque PvAT were associated with a higher risk of coronary thrombosis and were correlated with histological components of plaque progression and destabilization. M2 macrophages were correlated with plaque size, calcification, necrotic content, and a decrease in the number of vasa vasorum in the adventitia layer.

Activation of Complement Components on Circulating Blood Monocytes From COVID-19 Patients.

The coronavirus disease-2019 (COVID-19) caused by the SARS-CoV-2 virus may vary from asymptomatic to severe infection with multi-organ failure and death. Increased levels of circulating complement biomarkers have been implicated in COVID-19-related hyperinflammation and coagulopathy. We characterized systemic complement activation at a cellular level in 49-patients with COVID-19. We found increases of the classical complement sentinel C1q and the downstream C3 component on circulating blood monocytes from COVID-19 patients when compared to healthy controls (HCs). Interestingly, the cell surface-bound complement inhibitor CD55 was also upregulated in COVID-19 patient monocytes in comparison with HC cells. Monocyte membrane-bound C1q, C3 and CD55 levels were associated with plasma inflammatory markers such as CRP and serum amyloid A during acute infection. Membrane-bounds C1q and C3 remained elevated even after a short recovery period. These results highlight systemic monocyte-associated complement activation over a broad range of COVID-19 disease severities, with a compensatory upregulation of CD55. Further evaluation of complement and its interaction with myeloid cells at the membrane level could improve understanding of its role in COVID-19 pathogenesis.

Undernutrition and Cachexia in Patients with Decompensated Heart Failure and Chagas Cardiomyopathy: Occurrence and Association with Hospital Outcomes. / Desnutrição e Caquexia na Insuficiência Cardíaca Descompensada e Cardiomiopatia Chagásica: Ocorrência e Associação com Desfechos Hospitalares.

BACKGROUND: Nutritional disorders are common among patients with heart failure (HF) and associated with poor prognosis. Importantly, some populations of patients, like the ones with Chagas disease, are frequently excluded from most analyses. OBJECTIVE: We sought to study the occurrence of undernutrition and cachexia in patients with Chagas disease during episodes of decompensated HF (DHF) as compared to other etiologies, and to investigate the influence of these findings on hospital outcomes. METHODS: We performed a consecutive case series study with patients hospitalized with DHF. Patients underwent the Subjective Global Assessment of nutritional status (SGA), besides anthropometric and laboratorial measures, and were evaluated for the occurrence of cachexia, low muscle mass and strength. We studied the occurrence of death or urgent heart transplantation during hospitalization. RESULTS: Altogether, 131 patients were analyzed and 42 (32.1%) had Chagas disease. Patients with Chagas disease had lower Body Mass Index (BMI) (22.4 kg/m2[19.9-25.3] vs. 23.6 kg/m2 [20.8-27.3], p=0.03), higher frequency of undernutrition (76.2% vs 55.1%, p=0.015) and higher occurrence of death or transplant (83.3% vs. 41.6%, p<0.001). We found that, in patients with Chagas etiology, the occurrence of death or cardiac transplantation were associated with undernutrition (3 [42.9%] patients with hospital discharge vs 29 [82.9%] patients with death or heart transplant, p=0.043). CONCLUSIONS: Taken together, our results indicate that patients with Chagas disease hospitalized with DHF often present with nutritional disorders, especially undernutrition; importantly, this finding was associated with the occurrence of death and heart transplant during hospitalization.

FUNDAMENTO: Problemas nutricionais são comuns em pacientes com insuficiência cardíaca (IC) e estão associados a um prognóstico ruim. É relevante mencionar que algumas populações de pacientes, como os com Doença de Chagas, são normalmente excluídas da maioria das análises. OBJETIVO: Buscamos analisar a ocorrência de desnutrição e caquexia em pacientes com Doença de Chagas durante episódios de IC descompensada (ICD) em comparação a outras etiologias, e investigar a influência desses achados em desfechos hospitalares. MÉTODO: Realizamos um estudo de série de casos consecutivos com pacientes hospitalizados com ICD. Os pacientes foram submetidos à Avaliação Nutricional Subjetiva Global (ASG), além de medidas antropométricas e laboratoriais, e foram avaliados para a ocorrência de caquexia, baixa massa muscular e força. Estudamos a ocorrência de morte e transplante cardíaco de urgência durante a internação. RESULTADOS: Ao todo, 131 pacientes foram analisados e 42 (32,1%) tinham Doença de Chagas. Pacientes com Doença de Chagas apresentavam índice de massa corporal (IMC) menor (22,4 kg/m2 [19,9-25,3] vs. 23,6 kg/m2 [20,8-27,3], p=0,03), maior frequência de desnutrição (76,2% vs 55,1%, p=0,015) e mais ocorrências de morte ou transplante (83,3% vs. 41,6%, p<0,001). Observamos que, dentre os pacientes com etiologia da Doença de Chagas, a ocorrência de morte ou transplante cardíaco esteve associada com desnutrição (3 [42,9%] pacientes com alta hospitalar vs. 29 [82,9%] pacientes que morreram ou receberam transplante cardíaco, P=0,043). CONCLUSÕES: Ao todo, nossos resultados indicam que pacientes com Doença de Chagas internados com ICD costumam apresentar problemas nutricionais, principalmente desnutrição. É importante mencionar que este achado esteve associado à ocorrência de morte e transplante cardíaco durante a internação.

Desnutrição e Caquexia na Insuficiência Cardíaca Descompensada e Cardiomiopatia Chagásica: Ocorrência e Associação com Desfechos Hospitalares / Undernutrition and Cachexia in Patients with Decompensated Heart Failure and Chagas Cardiomyopathy: Occurrence and Association with Hospital Outcomes

Resumo Fundamento Problemas nutricionais são comuns em pacientes com insuficiência cardíaca (IC) e estão associados a um prognóstico ruim. É relevante mencionar que algumas populações de pacientes, como os com Doença de Chagas, são normalmente excluídas da maioria das análises. Objetivo Buscamos analisar a ocorrência de desnutrição e caquexia em pacientes com Doença de Chagas durante episódios de IC descompensada (ICD) em comparação a outras etiologias, e investigar a influência desses achados em desfechos hospitalares. Método Realizamos um estudo de série de casos consecutivos com pacientes hospitalizados com ICD. Os pacientes foram submetidos à Avaliação Nutricional Subjetiva Global (ASG), além de medidas antropométricas e laboratoriais, e foram avaliados para a ocorrência de caquexia, baixa massa muscular e força. Estudamos a ocorrência de morte e transplante cardíaco de urgência durante a internação. Resultados Ao todo, 131 pacientes foram analisados e 42 (32,1%) tinham Doença de Chagas. Pacientes com Doença de Chagas apresentavam índice de massa corporal (IMC) menor (22,4 kg/m2 [19,9-25,3] vs. 23,6 kg/m2 [20,8-27,3], p=0,03), maior frequência de desnutrição (76,2% vs 55,1%, p=0,015) e mais ocorrências de morte ou transplante (83,3% vs. 41,6%, p<0,001). Observamos que, dentre os pacientes com etiologia da Doença de Chagas, a ocorrência de morte ou transplante cardíaco esteve associada com desnutrição (3 [42,9%] pacientes com alta hospitalar vs. 29 [82,9%] pacientes que morreram ou receberam transplante cardíaco, P=0,043). Conclusões Ao todo, nossos resultados indicam que pacientes com Doença de Chagas internados com ICD costumam apresentar problemas nutricionais, principalmente desnutrição. É importante mencionar que este achado esteve associado à ocorrência de morte e transplante cardíaco durante a internação.

Abstract Background Nutritional disorders are common among patients with heart failure (HF) and associated with poor prognosis. Importantly, some populations of patients, like the ones with Chagas disease, are frequently excluded from most analyses. Objective We sought to study the occurrence of undernutrition and cachexia in patients with Chagas disease during episodes of decompensated HF (DHF) as compared to other etiologies, and to investigate the influence of these findings on hospital outcomes. Methods We performed a consecutive case series study with patients hospitalized with DHF. Patients underwent the Subjective Global Assessment of nutritional status (SGA), besides anthropometric and laboratorial measures, and were evaluated for the occurrence of cachexia, low muscle mass and strength. We studied the occurrence of death or urgent heart transplantation during hospitalization. Results Altogether, 131 patients were analyzed and 42 (32.1%) had Chagas disease. Patients with Chagas disease had lower Body Mass Index (BMI) (22.4 kg/m2[19.9-25.3] vs. 23.6 kg/m2 [20.8-27.3], p=0.03), higher frequency of undernutrition (76.2% vs 55.1%, p=0.015) and higher occurrence of death or transplant (83.3% vs. 41.6%, p<0.001). We found that, in patients with Chagas etiology, the occurrence of death or cardiac transplantation were associated with undernutrition (3 [42.9%] patients with hospital discharge vs 29 [82.9%] patients with death or heart transplant, p=0.043). Conclusions Taken together, our results indicate that patients with Chagas disease hospitalized with DHF often present with nutritional disorders, especially undernutrition; importantly, this finding was associated with the occurrence of death and heart transplant during hospitalization.

Rapid Emergence of T Follicular Helper and Germinal Center B Cells Following Antiretroviral Therapy in Advanced HIV Disease.

Low nadir CD4 T-cell counts in HIV+ patients are associated with high morbidity and mortality and lasting immune dysfunction, even after antiretroviral therapy (ART). The early events of immune recovery of T cells and B cells in severely lymphopenic HIV+ patients have not been fully characterized. In a cohort of lymphopenic (CD4 T-cell count < 100/µL) HIV+ patients, we studied mononuclear cells isolated from peripheral blood (PB) and lymph nodes (LN) pre-ART (n = 40) and 6-8 weeks post-ART (n = 30) with evaluation of cellular immunophenotypes; histology on LN sections; functionality of circulating T follicular helper (cTfh) cells; transcriptional and B-cell receptor profile on unfractionated LN and PB samples; and plasma biomarker measurements. A group of 19 healthy controls (HC, n = 19) was used as a comparator. T-cell and B-cell lymphopenia was present in PB pre-ART in HIV+ patients. CD4:CD8 and CD4 T- and B-cell PB subsets partly normalized compared to HC post-ART as viral load decreased. Strikingly in LN, ART led to a rapid decrease in interferon signaling pathways and an increase in Tfh, germinal center and IgD-CD27- B cells, consistent with histological findings of post-ART follicular hyperplasia. However, there was evidence of cTfh cells with decreased helper capacity and of limited B-cell receptor diversification post-ART. In conclusion, we found early signs of immune reconstitution, evidenced by a surge in LN germinal center cells, albeit limited in functionality, in HIV+ patients who initiate ART late in disease.

Classical complement and inflammasome activation converge in CD14highCD16- monocytes in HIV associated TB-immune reconstitution inflammatory syndrome.

Inflammasome-derived cytokines, IL-1ß and IL-18, and complement cascade have been independently implicated in the pathogenesis of tuberculosis (TB)-immune reconstitution inflammatory syndrome (TB-IRIS), a complication affecting HIV+ individuals starting antiretroviral therapy (ART). Although sublytic deposition of the membrane attack complex (MAC) has been shown to promote NLRP3 inflammasome activation, it is unknown whether these pathways may cooperatively contribute to TB-IRIS. To evaluate the activation of inflammasome, peripheral blood mononuclear cells (PBMCs) from HIV-TB co-infected patients prior to ART and at the IRIS or equivalent timepoint were incubated with a probe used to assess active caspase-1/4/5 followed by screening of ASC (apoptosis-associated speck-like protein containing a CARD domain) specks as a readout of inflammasome activation by imaging flow cytometry. We found higher numbers of monocytes showing spontaneous caspase-1/4/5+ASC-speck formation in TB-IRIS compared to TB non-IRIS patients. Moreover, numbers of caspase-1/4/5+ASC-speck+ monocytes positively correlated with IL-1ß/IL-18 plasma levels. Besides increased systemic levels of C1q and C5a, TB-IRIS patients also showed elevated C1q and C3 deposition on monocyte cell surface, suggesting aberrant classical complement activation. A clustering tSNE analysis revealed TB-IRIS patients are enriched in a CD14highCD16- monocyte population that undergoes MAC deposition and caspase-1/4/5 activation compared to TB non-IRIS patients, suggesting complement-associated inflammasome activation during IRIS events. Accordingly, PBMCs from patients were more sensitive to ex-vivo complement-mediated IL-1ß secretion than healthy control cells in a NLRP3-dependent manner. Therefore, our data suggest complement-associated inflammasome activation may fuel the dysregulated TB-IRIS systemic inflammatory cascade and targeting this pathway may represent a novel therapeutic approach for IRIS or related inflammatory syndromes.

The course of patients with Chagas heart disease during episodes of decompensated heart failure.

AIMS: This study aimed to analyse the clinical presentation and prognosis of patients with Chagas cardiomyopathy and decompensated heart failure (HF), as compared with other aetiologies. METHODS AND RESULTS: A prospective cohort of patients admitted with decompensated HF. We included 767 patients (63.9% male), with median age of 58 years [interquartile range 48.2-66.7 years]. Main aetiologies were non-Chagas/non-ischaemic cardiomyopathies in 389 (50.7%) patients, ischaemic disease in 209 (27.2%), and Chagas disease in 169 (22%). Median left ventricular ejection fraction was 26% (interquartile range 22-35%). Patients with Chagas differed from both patients with non-Chagas/non-ischaemic and ischaemic cardiomyopathies for a higher proportion of cardiogenic shock at admission (17.8%, 11.6%, and 11%, respectively, P < 0.001) and had lower blood pressure at admission (systolic blood pressure 90 [80-102.5], 100 [85-110], and 100 [88.2-120] mmHg, P < 0.001) and lower heart rate (heart rate 71 [60-80], 87 [70-102], and 79 [64-96.5] b.p.m., P < 0.001). Further, patients with Chagas had higher serum BNP level (1544 [734-3148], 1061 [465-239], and 927 [369-1455] pg/mL, P < 0.001), higher serum bilirubin (1.4 [0.922.44], 1.2 [0.77-2.19], and 0.84 [0.49-1.45] mg/dL, P < 0.001), larger left ventricular diameter (68 [63-73], 67 [58-74], and 62 [56.8-68.3] mm, respectively, P < 0.001), lower left ventricular ejection fraction (25 [21-30]%, 26 [22-35]%, and 30 [25-38]%, P < 0.001), and a higher proportion of patients with right ventricular function (48.8%, 40.7%, and 25.9%, P < 0.001). Patients with Chagas disease were more likely to receive inotropes than patients with non-Chagas/non-ischaemic and ischaemic cardiomyopathies (77.5%, 67.5%, and 62.5%, respectively, P = 0.007) and also to receive intra-aortic balloon pumping (30.8%, 16.2%, and 10.5%, P < 0.001). Overall, the rates of death or urgent transplant were higher among patients with Chagas than in other aetiologies, a difference that was driven mostly due to increased rate of heart transplant during hospital admission (20.2%, 10.3%, and 8.1%). The prognosis of patients at 180 days after hospital admission was worse for patients with Chagas disease as compared with other aetiologies. In patients with Chagas, age [odds ratio (OR) = 0.934, confidence interval (CI)95% 0.901-0.982, P = 0.005], right ventricular dysfunction by echocardiography (OR = 2.68, CI95% 1.055-6.81, P = 0.016), and urea (OR = 1.009, CI95% 1.001-1.018, P = 0.038) were significantly associated with prognosis. CONCLUSIONS: Patients with Chagas cardiomyopathy and decompensated HF have a distinct clinical presentation and worse prognosis compared with other aetiologies.

Lost in Translation: Lack of CD4 Expression due to a Novel Genetic Defect.

CD4 expression identifies a subset of mature T cells primarily assisting the germinal center reaction and contributing to CD8+ T-cell and B-cell activation, functions, and longevity. Herein, we present a family in which a novel variant disrupting the translation-initiation codon of the CD4 gene resulted in complete loss of membrane and plasma soluble CD4 in peripheral blood, lymph node, bone marrow, skin, and ileum of a homozygous proband. This inherited CD4 knockout disease illustrates the clinical and immunological features of a complete deficiency of any functional component of CD4 and its similarities and differences with other clinical models of primary or acquired loss of CD4+ T cells. The first inherited loss of any functional component of CD4, including soluble CD4, is clinically distinct from any other congenital or acquired CD4 T-cell defect and characterized by compensatory changes in T-cell subsets and functional impairment of B cells, monocytes, and natural killer cells.

Persistent Oxidative Stress and Inflammasome Activation in CD14highCD16- Monocytes From COVID-19 Patients.

The poor outcome of the coronavirus disease-2019 (COVID-19), caused by SARS-CoV-2, is associated with systemic hyperinflammatory response and immunopathology. Although inflammasome and oxidative stress have independently been implicated in COVID-19, it is poorly understood whether these two pathways cooperatively contribute to disease severity. Herein, we found an enrichment of CD14highCD16- monocytes displaying inflammasome activation evidenced by caspase-1/ASC-speck formation in severe COVID-19 patients when compared to mild ones and healthy controls, respectively. Those cells also showed aberrant levels of mitochondrial superoxide and lipid peroxidation, both hallmarks of the oxidative stress response, which strongly correlated with caspase-1 activity. In addition, we found that NLRP3 inflammasome-derived IL-1ß secretion by SARS-CoV-2-exposed monocytes in vitro was partially dependent on lipid peroxidation. Importantly, altered inflammasome and stress responses persisted after short-term patient recovery. Collectively, our findings suggest oxidative stress/NLRP3 signaling pathway as a potential target for host-directed therapy to mitigate early COVID-19 hyperinflammation and also its long-term outcomes.

Rab7 controls lipid droplet-phagosome association during mycobacterial infection.

Lipid droplets (LDs) are organelles that have multiple roles in inflammatory and infectious diseases. LD act as essential platforms for immunometabolic regulation, including as sites for lipid storage and metabolism, inflammatory lipid mediator production, and signaling pathway compartmentalization. Accumulating evidence indicates that intracellular pathogens may exploit host LDs as source of nutrients and as part of their strategy to promote immune evasion. Notably, numerous studies have demonstrated the interaction between LDs and pathogen-containing phagosomes. However, the mechanism involved in this phenomenon remains elusive. Here, we observed LDs and PLIN2 surrounding M. bovis BCG-containing phagosomes, which included observations of a bacillus cell surrounded by lipid content inside a phagosome and LAM from mycobacteria co-localizing with LDs; these results were suggestive of exchange of contents between these compartments. By using beads coated with M.tb lipids, we demonstrated that LD-phagosome associations are regulated through the mycobacterial cell wall components LAM and PIM. In addition, we demonstrated that Rab7 and RILP, but not Rab5, localizes to LDs of infected macrophages and observed the presence of Rab7 at the site of interaction with an infected phagosome. Moreover, treatment of macrophages with the Rab7 inhibitor CID1067700 significantly inhibited the association between LDs and LAM-coated beads. Altogether, our data demonstrate that LD-phagosome interactions are controlled by mycobacterial cell wall components and Rab7, which enables the exchange of contents between LDs and phagosomes and may represent a fundamental aspect of bacterial pathogenesis and immune evasion.

Evaluation of Canonical Inflammasome Activation in Human Monocytes by Imaging Flow Cytometry.

Canonical inflammasome activation is a tightly regulated process that has been implicated in a broad spectrum of inflammatory disorders. Inflammasome formation requires assembly of a cytosolic sensor protein with the adapter, ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain). Once formed, this multimeric protein structure allows for the activation of caspase-1, responsible for IL-1ß/IL-18 release. During this process, cytoplasmic dispersed ASC molecules cluster in one condensed micrometric-sized complex named ASC "speck," which is traditionally assessed by fluorescence microscopy and widely accepted as a readout for canonical inflammasome activation. However, equally reliable but less time-consuming quantitative methods have emerged as a significant need in order to improve clinical assessment of inflammasome-related conditions. Multispectral imaging flow cytometry (MIFC) combines the qualitative power of fluorescence microscopy with high throughput capabilities and multiplexing potential of flow cytometry into one single system. Here we explored the optimal imaging-based tools to measure ASC speck formation via imaging flow cytometry by using peripheral blood mononuclear cells (PBMCs) stimulated with the NLRP3 agonist Nigericin, as a positive control. We demonstrate that this technique is also able to detect the distribution of active caspase-1 within the ASC aggregates by incubating cells with FAM-FLICATM, a fluorochrome inhibitor of caspase-1. By applying these tools in PBMCs from patients with distinct inflammatory disorders we demonstrate that MIFC is able to assess canonical inflammasome activation in a quantitative and statistically robust manner in clinically relevant samples. Therefore, we propose that accurate assessment of specks by MIFC could help guide preventive or therapeutic strategies in an array of human inflammatory diseases in which inflammasomes play an important role.

Identification of rare HIV-1-infected patients with extreme CD4+ T cell decline despite ART-mediated viral suppression.

BACKGROUND: The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here, we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS: EXID's clinical and immunological characteristics were compared to immunological responders (IRs), immunological nonresponders (INRs), healthy controls (HCs), and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS: EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/µl, compared with CD4+ increases of 193 cells/µl and 427 cells/µl in INR and IR, respectively. EXID had reduced naive CD4+ T cells, but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR, but the IL-7 axis was profoundly perturbed compared with HC, IR, INR, and ICL. Genes involved in T cell and monocyte/macrophage function, autophagy, and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC, while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS: EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID.

Increased Metabolic Activity on 18F-Fluorodeoxyglucose Positron Emission Tomography-Computed Tomography in Human Immunodeficiency Virus-Associated Immune Reconstitution Inflammatory Syndrome.

Background: Immune reconstitution inflammatory syndrome (IRIS) represents an unexpected inflammatory response shortly after initiation of antiretroviral therapy (ART) in some human immunodeficiency virus (HIV)-infected patients with underlying neoplasia or opportunistic infections, including tuberculosis. We hypothesized that IRIS is associated with increased glycolysis and that 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET/CT) could help identify high-risk subjects. Methods: In this prospective cohort study, 30 HIV-infected patients (CD4+ count <100 cells/µL) underwent FDG-PET/CT scans at baseline and 4-8 weeks after ART initiation. Ten patients developed IRIS (6 mycobacterial). Results: At baseline, total glycolytic activity, total lesion volume, and maximum standardized uptake values (SUVs) of pathologic FDG uptake (reflective of opportunistic disease burden) were significantly higher in IRIS vs non-IRIS (P = .010, .017, and .029, respectively) and significantly correlated with soluble inflammatory biomarkers (interferon-γ, myeloperoxidase, tumor necrosis factor, interleukin 6, soluble CD14). Baseline bone marrow (BM) and spleen FDG uptake was higher in mycobacterial IRIS specifically. After ART initiation, BM and spleen mean SUV decreased in non-IRIS (P = .004, .013) but not IRIS subjects. Our results were supported by significantly higher glucose transporter 1 (Glut-1) expression of CD4+ cells and monocytes after ART initiation in IRIS/mycobacterial IRIS compared with non-IRIS patients. Conclusions: We conclude that increased pathologic metabolic activity on FDG-PET/CT prior to ART initiation is associated with IRIS development and correlates with inflammatory biomarkers. Abnormally elevated BM and spleen metabolism is associated with mycobacterial IRIS, HIV viremia, and Glut-1 expression on CD4+ cells and monocytes. Clinical Trials Registration: NCT02147405.

Cerebral blood flow changes during intermittent acute hypoxia in patients with heart failure.

Objective Heart failure (HF) is associated with intermittent hypoxia, and the effects of this hypoxia on the cardiovascular system are not well understood. This study was performed to compare the effects of acute hypoxia (10% oxygen) between patients with and without HF. Methods Fourteen patients with chronic HF and 17 matched control subjects were enrolled. Carotid artery changes were examined during the first period of hypoxia, and brachial artery changes were examined during the second period of hypoxia. Data were collected at baseline and after 2 and 4 minutes of hypoxia. Norepinephrine, epinephrine, dopamine, and renin were measured at baseline and after 4 minutes hypoxia. Results The carotid blood flow, carotid systolic diameter, and carotid diastolic diameter increased and the carotid resistance decreased in patients with HF. Hypoxia did not change the carotid compliance, distensibility, brachial artery blood flow and diameter, or concentrations of sympathomimetic amines in patients with HF, but hypoxia increased the norepinephrine level in the control group. Hypoxia increased minute ventilation and decreased the oxygen saturation and end-tidal carbon dioxide concentration in both groups. Conclusion Hypoxia-induced changes in the carotid artery suggest an intensification of compensatory mechanisms for preservation of cerebral blood flow in patients with HF.