Identification of kinase modulators as host-directed therapeutics against intracellular methicillin-resistant Staphylococcus aureus.

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.

CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

Distinct cellular immune profiles in the airways and blood of critically ill patients with COVID-19.

BACKGROUND: Knowledge of the pathophysiology of COVID-19 is almost exclusively derived from studies that examined the immune response in blood. We here aimed to analyse the pulmonary immune response during severe COVID-19 and to compare this with blood responses. METHODS: This was an observational study in patients with COVID-19 admitted to the intensive care unit (ICU). Mononuclear cells were purified from bronchoalveolar lavage fluid (BALF) and blood, and analysed by spectral flow cytometry; inflammatory mediators were measured in BALF and plasma. FINDINGS: Paired blood and BALF samples were obtained from 17 patients, four of whom died in the ICU. Macrophages and T cells were the most abundant cells in BALF, with a high percentage of T cells expressing the ƴδ T cell receptor. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells (87·3% and 83·8%, respectively), and these cells expressed higher levels of the exhaustion marker programmad death-1 than in peripheral blood. Prolonged ICU stay (>14 days) was associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. INTERPRETATION: The bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. Fully elucidating COVID-19 pathophysiology will require investigation of the pulmonary immune response.

Inhibition of Dendritic Cell Activation and Modulation of T Cell Polarization by the Platelet Secretome.

Platelet transfusions are a frequently administered therapy for especially hemato-oncological patients with thrombocytopenia. Next to their primary function in hemostasis, currently there is increased attention for the capacity of platelets to affect the function of various cells of the immune system. Here, we investigate the capacity of platelets to immuno-modulate monocyte-derived dendritic cells (moDC) as well as primary dendritic cells and effects on subsequent T cell responses. Platelets significantly inhibited pro-inflammatory (IL-12, IL-6, TNFα) and increased anti-inflammatory (IL-10) cytokine production of moDCs primed with toll-like receptor (TLR)-dependent and TLR-independent stimuli. Transwell assays and ultracentrifugation revealed that a soluble factor secreted by platelets, but not microvesicles, inhibited DC activation. Interestingly, platelet-derived soluble mediators also inhibited cytokine production by human ex vivo stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFNγ+ Th1 phenotype by moDCs. Overall, these results show that platelets are able to inhibit the pro-inflammatory properties of DCs, and may even induce an anti-inflammatory DC phenotype, with decreased T cell priming capacity by the DC. The results of this study provide more insight in the potential role of platelets in immune modulation, especially in the context of platelet transfusions.

Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes.

Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4+ T-cell response. Lastly, although the "don't eat me" signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.

The role of pathogen-reduced platelet transfusions on HLA alloimmunization in hemato-oncological patients.

BACKGROUND: Platelet transfusions can induce alloimmunization against HLA antigens. The use of pathogen-reduced platelet concentrates (PCs) was suggested to reduce HLA alloimmunization and concomitant transfusion refractoriness. METHODS: This study investigated HLA alloimmunization in available samples from 448 hemato-oncological patients who were randomized for the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial to receive either untreated or pathogen-reduced PCs (Mirasol, Terumo BCT Inc.). Anti-HLA Class I and II antibodies were determined before the first platelet transfusion and weekly thereafter using multiplex assay with standard cutoffs to detect low- as well as high-level antibodies. RESULTS: When using the lower cutoff, in patients who were antibody negative at enrollment, 5.4% (n = 12) developed anti-HLA Class I antibodies after receiving untreated PCs, while this was significantly higher in patients receiving pathogen-reduced PCs, 12.8% (n = 29; p = 0.009, intention-to-treat [ITT] analysis). A similar but nonsignificant trend was observed in the per-protocol (PP) analysis (5.4% vs. 10.1%; p = 0.15). HLA class II antibody formation was similar between both types of PCs in the ITT analysis, while the PP analysis showed a trend toward lower immunization after receiving pathogen-reduced PCs. Multivariate analysis identified receiving pathogen-reduced platelets as an independent risk factor for HLA Class I alloimmunization (ITT: odds ratio [95% confidence interval] = 3.02 [1.42-6.51], PP: odds ratio [95% confidence interval] = 2.77 [1.00-5.40]), without affecting HLA Class II alloimmunization. When using the high cutoff value, the difference in HLA Class I alloimmunization between study arms remained significant in the ITT analysis and again was not significant in the PP analysis. CONCLUSION: Our data clearly indicate that Mirasol pathogen inactivation does not prevent HLA Class I or II alloimmunization after platelet transfusions.

The quality of platelet concentrates related to corrected count increment: linking in vitro to in vivo.

BACKGROUND: Storage of platelet concentrates (PCs) results in reduced recovery and survival of transfused platelets (PLTs). Upon storage PLTs develop storage lesion that can be monitored by several laboratory tests. However, correlation of these descriptive tests with corrected count increments (CCIs), a marker frequently used to establish the effectiveness of PLT transfusions, is limited or unknown. This study investigated to what extent a functional test or a combined in vitro rating score improves the correlation of laboratory tests with 1-hour CCI. STUDY DESIGN AND METHODS: PCs were analyzed using six different laboratory tests (n = 123) before transfusion in a prophylactic setting to 74 hematooncologic patients. Linear regression and Spearman correlation were used to determine associations between descriptive (either separately or combined in an in vitro rating score) or functional test results and 1-hour CCIs obtained after transfusion. RESULTS: CD62P expression (r = -0.45), annexin V binding (r = -0.36), the updated in vitro rating score (r = 0.50), and PLT responsiveness after thrombin receptor activator for peptide-6 (TRAP) (r = 0.43-0.57) or adenosine diphosphate stimulation (r = 0.11-0.51) significantly correlated to 1-hour CCIs obtained after transfusion, whereas lactate concentration, ThromboLUX score, and thromboelastography measurements did not. The strongest correlations were observed for in vitro rating score and PLT responsiveness after TRAP stimulation and these tests could explain 24 and 33% of the observed variation in 1-hour CCI, respectively. CONCLUSION: Combining descriptive markers in one in vitro rating score improved correlation to 1-hour CCI compared to the tests separately. Of all tests investigated, mean PLT responsiveness after TRAP stimulation showed the strongest clinical correlation and was best able to predict the 1-hour CCI.

Platelets from donors with consistently low HLA-B8, -B12, or -B35 expression do not undergo antibody-mediated internalization.

Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.