During systemic inflammation different neutrophil subsets are mobilized to the peripheral blood. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16^bright/CD62L^bright), based on either an immature CD16^dim/CD62L^bright or a CD16^bright/CD62L^dim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but how neutrophils become suppressive is unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes in mRNA expression that are relevant for their functions. Neutrophil subsets were isolated by fluorescence-activated cell sorting from blood of healthy volunteers that were administered a single dose of lipopolysaccharide (2 ng/kg i.v.) and the transcriptome was determined by microarray analysis. Interestingly, the CD16^bright/CD62L^dim suppressive neutrophils showed an interferon-induced transcriptome profile. More importantly, IFN-γ, but not IFN-α or IFN-β stimulated neutrophils, acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that IFN-γ-induced expression of PD-L1 on neutrophils enables suppression of lymphocyte proliferation. Specific stimulation of neutrophils present at the inflammatory sites might therefore have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease.

The incidence of severe Haemophilus influenza infections, such as sepsis and meningitis, has declined substantially since the introduction of the H. influenzae serotype b vaccine. However, the H. influenzae type b vaccine fails to protect against nontypeable H. influenzae strains, which have become increasingly frequent causes of invasive disease, especially among children and the elderly. We summarize recent literature supporting the emergence of invasive nontypeable H. influenzae and describe mechanisms that may explain its increasing prevalence over the past 2 decades.

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept. Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Long-lasting and sterile homologous protection against malaria can be achieved by the exposure of malaria-naive volunteers under chemoprophylaxis to Plasmodium falciparum -infected mosquitoes (chemoprophylaxis and sporozoite [CPS] immunization). While CPS-induced antibodies neutralize sporozoite infectivity in vitro and in vivo , antibody-mediated effector mechanisms are still poorly understood. Here, we investigated whether complement contributes to CPS-induced preerythrocytic immunity. Sera collected before and after CPS immunization in the presence of active or inactive complement were assessed for the recognition of homologous NF54 and heterologous NF135.C10 sporozoites, complement fixation, sporozoite lysis, and possible subsequent effects on in vitro sporozoite infectivity in human hepatocytes. CPS immunization induced sporozoite-specific IgM ( P < 0.0001) and IgG ( P = 0.001) antibodies with complement-fixing capacities ( P < 0.0001). Sporozoite lysis ( P = 0.017), traversal ( P < 0.0001), and hepatocyte invasion inhibition ( P < 0.0001) by CPS-induced antibodies were strongly enhanced in the presence of active complement. Complement-mediated invasion inhibition in the presence of CPS-induced antibodies negatively correlated with cumulative parasitemia during CPS immunizations ( P = 0.013). While IgG antibodies similarly recognized homologous and heterologous sporozoites, IgM binding to heterologous sporozoites was reduced ( P = 0.023). Although CPS-induced antibodies did not differ in their abilities to fix complement, lyse sporozoites, or inhibit the traversal of homologous and heterologous sporozoites, heterologous sporozoite invasion was more strongly inhibited in the presence of active complement ( P = 0.008). These findings demonstrate that CPS-induced antibodies have complement-fixing activity, thereby significantly further enhancing the functional inhibition of homologous and heterologous sporozoite infectivity in vitro . The combined data highlight the importance of complement as an additional immune effector mechanism in preerythrocytic immunity after whole-parasite immunization against Plasmodium falciparum malaria.

The immune inhibitory checkpoint molecule programmed death ligand (PD-L)-1 is increasingly recognized as an important player in the immune suppression observed in patients with sepsis, but its role has mainly been studied in monocytes. In an earlier study, we demonstrated that experimental human endotoxemia results in mobilization of a subset of PD-L1-expressing neutrophils displaying an IFN-γ-induced transcriptome profile. Herein, we identify the source of IFN-γ production during murine endotoxemia and its role in the generation of PD-L1⁺-suppressive neutrophils. We demonstrate that, similar to what we found in humans, murine endotoxemia results in the influx of a subset of PD-L1⁺ neutrophils in the circulation, and incubation of mouse neutrophils with recombinant IFN-γ profoundly increases PD-L1 expression. Furthermore, administration of anti-IFN-γ abrogated the generation of PD-L1⁺ neutrophils in endotoxemic mice. The critical involvement of the spleen is illustrated by the fact that splenectomy nullified circulating IFN-γ levels and substantially reduced the abundance of PD-L1⁺ neutrophils, whereas cotreatment with recombinant IFN-γ resulted in complete restoration of generation of PD-L1⁺ neutrophils in splenectomized mice. Finally, the functional importance of spleen-derived PD-L1⁺ neutrophils is exemplified by the finding that the profound decrease in T-lymphocyte proliferation observed in cells from endotoxemic mice was attenuated in cells from splenectomized animals. We demonstrated that spleen-derived IFN-γ induces generation of PD-L1⁺-suppressive neutrophils, implying that the spleen is critically involved in immune suppression during inflammatory diseases such as sepsis. Furthermore, our data suggest that IFN-γ plays a dual role by enhancing innate immunity and at the same time suppressing adaptive immune responses.

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.