NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins.

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir.

Human immunodeficiency virus (HIV) infection induces immunological dysfunction, which limits the elimination of HIV-infected cells during treated infection. Identifying and targeting dysfunctional immune cells might help accelerate the purging of the persistent viral reservoir. Here, we show that chronic HIV infection increases natural killer (NK) cell populations expressing the negative immune regulator KLRG1, both in peripheral blood and lymph nodes. Antiretroviral treatment (ART) does not reestablish these functionally impaired NK populations, and the expression of KLRG1 correlates with active HIV transcription. Targeting KLRG1 with specific antibodies significantly restores the capacity of NK cells to kill HIV-infected cells, reactivates latent HIV present in CD4+ T cells co-expressing KLRG1, and reduces the intact HIV genomes in samples from ART-treated individuals. Our data support the potential use of immunotherapy against the KLRG1 receptor to impact the viral reservoir during HIV persistence.

NLRC4-mediated activation of CD1c+ DC contributes to perpetuation of synovitis in rheumatoid arthritis.

The individual contribution of specific myeloid subsets such as CD1c+ conventional DC (cDC) to perpetuation of rheumatoid arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDC in patients with RA and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional, and functional characteristics of CD1c+ and CD141+ cDC and monocytes from the blood and synovial fluid of patients with RA. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC was associated with the presence of this DC subset in the synovial membrane in patients with RA. Moreover, synovial CD1c+ cDC are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFN-γ+IL-17+CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ receptors and NLRC4 as a potential molecular mechanism mediating pathogenic activation, CD64 upregulation, and functional specialization of CD1c+ cDC in response to dsDNA-IgG in patients with RA.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV.

BACKGROUND: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. METHODS: We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. FINDINGS: HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. INTERPRETATION: Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. FUNDING: NIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients.

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.

IL-6 serum levels predict severity and response to tocilizumab in COVID-19: An observational study.

BACKGROUND: Patients with coronavirus disaese 2019 (COVID-19) can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome requiring invasive mechanical ventilation (IMV). Because IL-6 is a relevant cytokine in acute respiratory distress syndrome, the blockade of its receptor with tocilizumab (TCZ) could reduce mortality and/or morbidity in severe COVID-19. OBJECTIVE: We sought to determine whether baseline IL-6 serum levels can predict the need for IMV and the response to TCZ. METHODS: A retrospective observational study was performed in hospitalized patients diagnosed with COVID-19. Clinical information and laboratory findings, including IL-6 levels, were collected approximately 3 and 9 days after admission to be matched with preadministration and postadministration of TCZ. Multivariable logistic and linear regressions and survival analysis were performed depending on outcomes: need for IMV, evolution of arterial oxygen tension/fraction of inspired oxygen ratio, or mortality. RESULTS: One hundred forty-six patients were studied, predominantly males (66%); median age was 63 years. Forty-four patients (30%) required IMV, and 58 patients (40%) received treatment with TCZ. IL-6 levels greater than 30 pg/mL was the best predictor for IMV (odds ratio, 7.1; P < .001). Early administration of TCZ was associated with improvement in oxygenation (arterial oxygen tension/fraction of inspired oxygen ratio) in patients with high IL-6 (P = .048). Patients with high IL-6 not treated with TCZ showed high mortality (hazard ratio, 4.6; P = .003), as well as those with low IL-6 treated with TCZ (hazard ratio, 3.6; P = .016). No relevant serious adverse events were observed in TCZ-treated patients. CONCLUSIONS: Baseline IL-6 greater than 30 pg/mL predicts IMV requirement in patients with COVID-19 and contributes to establish an adequate indication for TCZ administration.

COVID-19 severity associates with pulmonary redistribution of CD1c+ DCs and inflammatory transitional and nonclassical monocytes.

SARS-CoV-2 is responsible for the development of coronavirus disease 2019 (COVID-19) in infected individuals, who can either exhibit mild symptoms or progress toward a life-threatening acute respiratory distress syndrome (ARDS). Exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. Here, we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood and lung of COVID-19 patients with different clinical severity in comparison with healthy individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Our results indicate that inflammatory transitional and nonclassical monocytes and CD1c+ conventional dendritic cells preferentially migrate from blood to lungs in patients with severe COVID-19. Thus, this study increases the knowledge of specific myeloid subsets involved in the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies for fighting SARS-CoV-2 infection.

Differential Redistribution of Activated Monocyte and Dendritic Cell Subsets to the Lung Associates with Severity of COVID-19.

The SARS-CoV-2 is responsible for the pandemic COVID-19 in infected individuals, who can either exhibit mild symptoms or progress towards a life-threatening acute respiratory distress syndrome (ARDS). It is known that exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. With the aim to improve the knowledge in this area, we developed a cross-sectional study, in which we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood of COVID-19 patients with different clinical severity in comparison with healthy control individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Collectively, our results suggest that inflammatory transitional and non-classical monocytes preferentially migrate from blood to lungs in patients with severe COVID-19. CD1c+ conventional dendritic cells also followed this pattern, whereas CD141+ conventional and CD123hi plasmacytoid dendritic cells were depleted from blood but were absent in the lungs. Thus, this study increases the knowledge on the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies to fight SARS-CoV-2 infection.