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.

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.

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.

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.

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.

Emerging Concepts about NAIP/NLRC4 Inflammasomes.

Neuronal apoptosis inhibitory protein (NAIP)/NOD-like receptor (NLR) containing a caspase activating and recruitment domain (CARD) 4 (NLRC4) inflammasome complexes are activated in response to proteins from virulent bacteria that reach the cell cytosol. Specific NAIP proteins bind to the agonists and then physically associate with NLRC4 to form an inflammasome complex able to recruit and activate pro-caspase-1. NAIP5 and NAIP6 sense flagellin, component of flagella from motile bacteria, whereas NAIP1 and NAIP2 detect needle and rod components from bacterial type III secretion systems, respectively. Active caspase-1 mediates the maturation and secretion of the pro-inflammatory cytokines, IL-1ß and IL-18, and is responsible for the induction of pyroptosis, a pro-inflammatory form of cell death. In addition to these well-known effector mechanisms, novel roles have been described for NAIP/NLRC4 inflammasomes, such as phagosomal maturation, activation of inducible nitric oxide synthase, regulation of autophagy, secretion of inflammatory mediators, antibody production, activation of T cells, among others. These effector mechanisms mediated by NAIP/NLRC4 inflammasomes have been extensively studied in the context of resistance of infections and the potential of their agonists has been exploited in therapeutic strategies to non-infectious pathologies, such as tumor protection. Thus, this review will discuss current knowledge about the activation of NAIP/NLRC4 inflammasomes and their effector mechanisms.