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Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defence. Recent studies have implicated these peptides in the clearance of aberrant cells and in neurodegenerative syndromes. In Drosophila, many AMPs are produced downstream of Toll and Imd NF-κB pathways upon infection. Upon aging, AMPs are upregulated, drawing attention to these molecules as possible causes of age-associated inflammatory diseases. However, functional studies overexpressing or silencing these genes have been inconclusive. Using an isogenic set of AMP gene deletions, we investigated the net impact of AMPs on aging. Overall, we found no major effect of individual AMPs on lifespan, with the possible exception of Defensin. However, ΔAMP14 flies lacking seven AMP gene families displayed reduced lifespan. Increased bacterial load in the food of aged ΔAMP14 flies suggested that their lifespan reduction was due to microbiome dysbiosis, consistent with a previous study. Moreover, germ-free conditions extended the lifespan of ΔAMP14 flies. Overall, our results did not point to an overt role of individual AMPs in lifespan. Instead, we found that AMPs collectively impact lifespan by preventing dysbiosis during aging.

Most studies of infections at mucosal surfaces have focused on the acute phase of the disease. Consequently, little is known about the molecular processes that underpin tissue recovery and the long-term consequences postinfection. Here, we conducted temporal deep quantitative proteomic analysis of colonic intestinal epithelial cells (cIECs) from mice infected with the natural mouse pathogen Citrobacter rodentium over time points corresponding to the late steady-state phase (10 days postinfection [DPI]), the clearance phase (13 to 20 DPI), and 4 weeks after the pathogen has been cleared (48 DPI). C. rodentium, which relies on a type III secretion system to infect, is used to model infections with enteropathogenic and enterohemorrhagic Escherichia coli. We observe a strong upregulation of inflammatory signaling and nutritional immunity responses during the clearance phase of the infection. Despite morphological tissue recovery, chromogranin B (ChgB)-positive endocrine cells remained significantly below baseline levels at 48 DPI. In contrast, we observed an increased abundance of proteins involved in antigen processing and presentation 4 weeks after pathogen clearance. In particular, long-term changes were characterized by a persistent interferon gamma (IFN-γ) response and the expression of major histocompatibility complex class II (MHCII) molecules in 60% of the EpCAM+ cIECs, which were not seen in Ifnγ−/− mice. Nonetheless, both wild-type and Ifnγ−/− mice mounted similar systemic and colonic IgG responses to C. rodentium and were equally protected from rechallenge, suggesting that cIEC MHCII is not necessary for protective immunity against C. rodentium.

Coronavirus 2 (SARS-CoV2) (COVID-19) causes severe acute respiratory syndrome. Severe illness of COVID-19 largely occurs in older people and recent evidence indicates that demented patients have higher risk for COVID-19. Additionally, COVID-19 further enhances the vulnerability of older adults with cognitive damage. A balance between the immune and inflammatory response is necessary to control the infection. Thus, antimicrobial and anti-inflammatory drugs are hopeful therapeutic agents for the treatment of COVID-19. Accumulating evidence suggests that lactoferrin (Lf) is active against SARS-CoV-2, likely due to its potent antiviral and anti-inflammatory actions that ultimately improves immune system responses. Remarkably, salivary Lf levels are significantly reduced in different Alzheimer's disease (AD) stages, which may reflect AD-related immunological disturbances, leading to reduced defense mechanisms against viral pathogens and an increase of the COVID-19 susceptibility. Overall, there is an urgent necessity to protect AD patients against COVID-19, decreasing the risk of viral infections. In this context, we propose bovine Lf (bLf) as a promising preventive therapeutic tool to minimize COVID-19 risk in patients with dementia or AD. This study was supported by grants from Instituto de Salud Carlos III (FIS21/00679 to EC and JH-G and PI21/00183 to FB), FEDER, Comunidad de Madrid (S2017/BMD-3700; NEUROMETAB-CM to EC), and CIBERNED (CB07/502 to EC); Spanish Ministry of Economy and Competitiveness (PID2020-119978RB-I00 to JC), CIBERNED, the Research Program for a Long-Life Society (0551_PSL_6_E to JC), the Junta de Andalucía (PY20_00858 to J.L.C.), the Andalucía-FEDER Program (UPO-1380913 to JC). Sí

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract. Funding: The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, Rockefeller University; the St. Giles Foundation; the NIH (R01AI088364), the National Center for Advancing Translational Sciences (NCATS); NIH Clinical and Translational Science Award (CTSA) program (UL1TR001866); a Fast Grant from Emergent Ventures; Mercatus Center at the George Mason University; the Yale Center for Mendelian Genomics and the GSP Coordinating Center funded by the National Human Genome Research Institute (NHGRI) (UM1HG006504 and U24HG008956); the Meyer Foundation; the JPB Foundation; the French National Research Agency (ANR) under the “Investments for the Future” program (ANR-10-IAHU-01) and the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID); the French Foundation for Medical Research (FRM) (EQU201903007798); the FRM and ANR GENCOVID project, the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AABIFNCOV (ANR-20-CO11-0001) projects; the European Union’s Horizon 2020 Research and Innovation Program under grant agreement no. 824110 (EASI-genomics). The French COVID Cohort study group was sponsored by INSERM and supported by the REACTing consortium and by a grant from the French Ministry of Health (PHRC 20-0424). The Cov-Contact Cohort was supported by the REACTing consortium, the French Ministry of Health, and the European Commission (RECOVER WP 6). The Neurometabolic Diseases Laboratory received funding from the European Union’s Horizon 2020 Research and Innovation Program (EasiGenomics grant no. 824110 COVID-19/PID12342). A.P., R.P.d.D., C.R.-G., and C.F. were funded by Instituto de Salud Carlos III (COV20_01333 and COV20_01334), the Spanish Ministry of Science and Innovation (RTC-2017-6471-1; AEI/FEDER, UE), Fundación DISA (OA18/017), and Cabildo Insular de Tenerife (CGIEU0000219140 and “Apuestas científicas del ITER para colaborar en la lucha contra la COVID-19”). The laboratories of G.N. and A.N. were supported by a grant awarded to Regione Lazio (PROGETTI DI GRUPPI DI RICERCA 2020) no. A0375-2020-36663, GecoBiomark. A. Amara’s laboratory was supported by ANR under the “Investments for the Future” program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the FRM (EQU202003010193), ANR (ANR-20-COVI-000 project IDISCOVR and ANR-20-CO11-0004 project FISHBP), and the University of Paris (Plan de Soutien Covid-19: RACPL20FIR01-COVID-SOUL). This work was supported, in part, by the Division of Intramural Research, NIAID, NIH (grants 1ZIAAI001265 to H.C.S. and ZIA AI001270 to L.D.N.). The G.C. laboratory was supported by the Italian Ministry of Health (grant COVID-2020-12371617) and the intramural COVID Host Genetics program. The J.L.F. laboratory was supported, in part, by the Coopération Scientifique France-Colciencias (ECOS-Nord/COLCIENCIAS/MEN/ICETEX; 806-2018) and Colciencias contract 713-2016 (no. 111574455633). The V.S. laboratory was supported by ANR DENDRISEPSIS (ANR-17-CE15-0003) and ANR APCOD (ANR-17-CE15-0003-01), a Fast Grant from the Mercatus Center, FRM, University of Paris PLAN D’URGENCE COVID19. The N.M. laboratory was supported by Sidra Medicine (SDR400048) and the Qatar National Research Fund (grant No. NPRP9-251-3-045)

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract. ispartof: SCIENCE IMMUNOLOGY vol:6 issue:62 ispartof: location:United States status: published

SARS-CoV-2 started spreading toward the end of 2019 causing COVID-19, a disease that reached pandemic proportions among the human population within months. The reasons for the spectrum of differences in the severity of the disease across the population, and in particular why the disease affects more severely the aging population and those with specific preconditions are unclear. We developed machine learning models to mine 240,000 scientific articles openly accessible in the CORD-19 database, and constructed knowledge graphs to synthesize the extracted information and navigate the collective knowledge in an attempt to search for a potential common underlying reason for disease severity. The machine-driven framework we developed repeatedly pointed to elevated blood glucose as a key facilitator in the progression of COVID-19. Indeed, when we systematically retraced the steps of the SARS-CoV-2 infection, we found evidence linking elevated glucose to each major step of the life-cycle of the virus, progression of the disease, and presentation of symptoms. Specifically, elevations of glucose provide ideal conditions for the virus to evade and weaken the first level of the immune defense system in the lungs, gain access to deep alveolar cells, bind to the ACE2 receptor and enter the pulmonary cells, accelerate replication of the virus within cells increasing cell death and inducing an pulmonary inflammatory response, which overwhelms an already weakened innate immune system to trigger an avalanche of systemic infections, inflammation and cell damage, a cytokine storm and thrombotic events. We tested the feasibility of the hypothesis by manually reviewing the literature referenced by the machine-generated synthesis, reconstructing atomistically the virus at the surface of the pulmonary airways, and performing quantitative computational modeling of the effects of glucose levels on the infection process. We conclude that elevation in glucose levels can facilitate the progression of the disease through multiple mechanisms and can explain much of the differences in disease severity seen across the population. The study provides diagnostic considerations, new areas of research and potential treatments, and cautions on treatment strategies and critical care conditions that induce elevations in blood glucose levels., Graphical Abstract Most of the patients at risk of severe COVID-19, present with high blood glucose levels or dysregulated glycemia. Subsequent increased concentrations of glucose in the ASL and intracellularly, provide ideal conditions for SARS-CoV-2 to evade the innate defence of the lungs and replicate in the pulmonary cells. High blood glucose also facilitates the hyper-inflammation observed in the cytokine storm. The subsequent high viral load (1) correlates with SARS-CoV-2 specific inactivation of ACE2 and increased concentration of AngII, that aggravates the dysregulation of the glycemic control in a vicious circle of viral infection (2). Finally, the combination of high blood glucose and overproduction of AngII (3) leads to the phenomenon involved in the complications observed in severe cases such as multi-organ failures and thrombotic events.

cited By 0 We describe a case of Lichtenberg Figures (LFs) following an electrical injury from a high-voltage switchgear in a 47 year-old electrician. LFs, also known as ferning pattern or keraunographic markings, are a pathognomonic skin sign for lightning strike injuries. Their true pathophysiology has remained a mystery and only once before described following an electical injury. The aim was to characterise the tissue response of LFs by performing untargeted non-labelled proteomics and immunohistochemistry on paraffin-embedded sections of skin biopsies taken from the area of LFs at presentation and at 3 months follow-up. Our results demonstrated an increase in dermal T-cells and greatly increased expression of the iron-binding glycoprotein lactoferrin by keratinocytes and lymphocytes. These changes in the LF-affected skin were associated with extravasation of red blood cells from dermal vessels. Our results provide an initial molecular and cellular insight into the tissue response associated with LFs.

Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the attachment and penetration of the plant surface. Appressoria, specialized infection structures, have been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline appressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appressoria, their initiation, their formation, and their function in fungi are discussed. We reviewed the molecular mechanisms regulating the formation and function of appressoria, their strategies to evade host defenses, and the related genomics and transcriptomics. The current review provides a foundation for comprehensive studies regarding their evolution and diversity in different fungal groups.

Pneumonia is a common acute respiratory infection that affects the alveoli and distal airways; it is a major health problem and associated with high morbidity and short-term and long-term mortality in all age groups worldwide. Pneumonia is broadly divided into community-acquired pneumonia or hospital-acquired pneumonia. A large variety of microorganisms can cause pneumonia, including bacteria, respiratory viruses and fungi, and there are great geographical variations in their prevalence. Pneumonia occurs more commonly in susceptible individuals, including children of

Summary Interferon-inducible guanylate-binding proteins (GBPs) promote cell-intrinsic defense through host cell death. GBPs target pathogens and pathogen-containing vacuoles and promote membrane disruption for release of microbial molecules that activate inflammasomes. GBP1 mediates pyroptosis or atypical apoptosis of Salmonella Typhimurium (STm)- or Toxoplasma gondii (Tg)- infected human macrophages, respectively. The pathogen-proximal detection-mechanisms of GBP1 remain poorly understood, as humans lack functional immunity-related GTPases (IRGs) that assist murine Gbps. Here, we establish that GBP1 promotes the lysis of Tg-containing vacuoles and parasite plasma membranes, releasing Tg-DNA. In contrast, we show GBP1 targets cytosolic STm and recruits caspase-4 to the bacterial surface for its activation by lipopolysaccharide (LPS), but does not contribute to bacterial vacuole escape. Caspase-1 cleaves and inactivates GBP1, and a cleavage-deficient GBP1D192E mutant increases caspase-4-driven pyroptosis due to the absence of feedback inhibition. Our studies elucidate microbe-specific roles of GBP1 in infection detection and its triggering of the assembly of divergent caspase signaling platforms., Graphical Abstract, Highlights • Development of two microscopy assays for microbe/microbe-containing vacuole lysis • Human GBP1 is essential for the lysis of Toxoplasma gondii vacuoles and parasites • Caspase-4 recruitment, but not cytosolic escape of Salmonella, is GBP1 dependent • Caspase-1 cleaves and inactivates GBP1 and suppresses caspase-4-driven pyroptosis, Fisch et al. find that GBP1 targets Toxoplasma vacuolar and parasite membranes for disruption of both membranes. In contrast, appearance of cytosolic Salmonella is GBP1 independent, but caspase-4 recruitment to bacteria and activation is GBP1 dependent. In a negative feedback loop, caspase-1 cleaves GBP1 and suppresses caspase-4-driven pyroptosis during Salmonella infection.

COVID-19 infection has a heterogenous disease course it may be asymptomatic or causes only mild symptoms in the majority of the cases, while immunologic complications such as macrophage activation syndrome also known as secondary hemophagocytic lymphohistiocytosis, resulting in cytokine storm syndrome and acute respiratory distress syndrome, may also occur in some patients. According to current literature, impairment of SARS-CoV-2 clearance due to genetic and viral features, lower levels of interferons, increased neutrophil extracellular traps, and increased pyroptosis and probable other unknown mechanisms create a background for severe disease course complicated by macrophage activation syndrome and cytokine storm. Various genetic mutations may also constitute a risk factor for severe disease course and occurrence of cytokine storm in COVID-19. Once, immunologic complications like cytokine storm occur, anti-viral treatment alone is not enough and should be combined with appropriate anti-inflammatory treatment. Anti-rheumatic drugs, which are tried for managing immunologic complications of COVID-19 infection, will also be discussed including chloroquine, hydroxychloroquine, JAK inhibitors, IL-6 inhibitors, IL-1 inhibitors, anti-TNF-alpha agents, corticosteroids, intravenous immunoglobulin (IVIG), and colchicine. Early recognition and appropriate treatment of immunologic complications will decrease the morbidity and mortality in COVID-19 infection, which requires the collaboration of infectious disease, lung, and intensive care unit specialists with other experts such as immunologists, rheumatologists, and hematologists.

BackgroundSaliva contains a very complex mixture of proteins for defense against microbiological pathogens and for oral food perception. Howler monkeys are Neotropical primates that can consume a mostly leaf diet. They are well known to thrive in highly disturbed habitats where they may cope with a diversity of dietary challenges and infection risks. We aimed to describe the salivary proteome of howlers to contribute to better understanding of their physiology.MethodsWe analyzed the salivary proteins of wild black howler monkeys (Alouatta pigra), by SDS-PAGE-1-D and Nano LC-MS/MS and categorized them by their function involved in host defense and oral food perception.ResultsOur proteomic analysis identified 156 proteins in howler saliva including a number of host defense peptides that are the first line of defense in mammals, such as defensin, cathelicidin, dermcidin, and lactotransferrin, and proteins with anti-bacterial, anti-fungal, and anti-viral capacity, such as IgA, IgG, IgM, BPI, salivary heat shock 70 kDa protein, beta-2-microbulin, and protein S-100. We also identified key proteins necessary for taste perception, including salivary carbonic anhydrase VI, cystatin D, IgA, and fatty acid-binding protein. Proteins to detect astringent foods were identifying, including four members of cystatins (A, B, C and D), lactoperoxidase, and histidine-rich proteins. No chitinase and amylase were identified as would be expected because howlers do not eat insects and little starch. These findings provide basic information to future studies in oral biology, ingestive physiology, and physiological ecology of mammals and non-human primates.

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor kappa B (NF-kappa B) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.

Background-Lipopolysaccharide (LPS) decreases hepatic CETP (cholesteryl ester transfer protein) expression albeit that the underlying mechanism is disputed. We recently showed that plasma CETP is mainly derived from Kupffer cells (KCs). In this study, we investigated the role of KC subsets in the mechanism by which LPS reduces CETP expression. Methods and Results-In CETP-transgenic mice, LPS markedly decreased hepatic CETP expression and plasma CETP concentration without affecting hepatic macrophage number. This was paralleled by decreased expression of the resting KC markers C-type lectin domain family 4, member f (Clec4f) and V-set and immunoglobulin domain containing 4 (Vsig4), while expression of the infiltrating monocyte marker lymphocyte antigen 6 complex locus C (Ly6C) was increased. Simultaneously, the ratio of plasma high-density lipoprotein-cholesterol over non-high-density lipoprotein-cholesterol transiently increased. After ablation hepatic macrophages via injection with liposomal clodronate, the reappearance of hepatic gene and protein expression of CETP coincided with Clec4f and Vsig4, but not Ly6C. Double-immunofluorescence staining showed that CETP co-localized with Clec4f(+)KCs and not Ly6C(+) monocytes. In humans, microarray gene-expression analysis of liver biopsies revealed that hepatic expression and plasma level of CETP both correlated with hepatic VSIG4 expression. LPS administration decreased the plasma CETP concentration in humans. In vitro experiments showed that LPS reduced liver X receptor-mediated CETP expression. Conclusions-Hepatic expression of CETP is exclusively confined to the resting KC subset (ie, F4/80(+)Clec4f(+)Vsig4(+)Ly6C(-)). LPS activated resting KCs, leading to reduction of Clec4f and Vsig4 expression and reduction of hepatic CETP expression, consequently decreasing plasma CETP and raising high-density lipoprotein (HDL)-cholesterol. This sequence of events is consistent with the anti-inflammatory role of HDL in the response to LPS and may be relevant as a defense mechanism against bacterial infections.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response., Author summary The ways that animals combat infection involve complex molecular pathways that are triggered upon microbial challenge. While a great deal is known about which pathways are key to a successful defence response, far less is known about exactly what elements of that response are critical to combat a given infection. Using the fruit fly–a genetic workhorse of Biology–we recently showed that a class of host-encoded antibiotics called “antimicrobial peptides” are essential for defence against bacterial infection, but do not contribute as strongly to defence against fungi. However a number of fly immune peptides remain uncharacterized, possibly explaining this gap in our understanding of the fly antifungal defence. Here we describe a novel antifungal peptide gene of fruit flies, and show that it is a major contributor to the fly antifungal defence response. We also found that this gene seems to regulate a behaviour that flies perform after infection, paralleling exciting recent findings that these genes are involved in neurological processes. Collectively, we clarify a key part of the fly antifungal defence, and contribute an important piece to help explain the logical organization of the immune defence against microbial infection.

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics. We would like to thank Sandra Delgado for the technical help in the preparation of the cryoEM vitrified grids and Dr. Isabel Uson and Dr. Ivan De Marino for the Arcimboldo software and valuable help. Funding was provided by the Unidad de Biofisica and the IKERBASQUE and MINECO science foundations.

OBJECTIVE Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury. DESIGN In vivo study. SETTING University research laboratory. SUBJECTS Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls. INTERVENTIONS Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes. MEASUREMENTS AND MAIN RESULTS Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells. CONCLUSIONS Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4–7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface., Author summary Invasive fungal infections remain an important health problem in immunocompromised patients. Immune recognition of fungal pathogens involves binding of specific cell wall components by pathogen recognition receptors (PRRs) and subsequent activation of immune defences. Some cell wall components are conserved among fungal species while other components are species-specific and phenotypically diverse. The fungal cell wall is dynamic and capable of changing its composition and organization when adapting to different growth niches and environmental stresses. Differences in the composition of the cell wall lead to differential immune recognition by the host. Understanding how changes in the cell wall composition affect recognition by PRRs is likely to be of major diagnostic and clinical relevance. Here we address this fundamental question using four soluble immune receptor-probes which recognize mannans and β-glucan in the cell wall. We use this novel methodology to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces was not correlated with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes on fungal cell surfaces are differentially distributed within and between the cell walls of fungal pathogens.

The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system., Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Life history strategies often shape biological interactions by specifying the parameters for possible encounters, such as the timing, frequency, or way of exposure to parasites. Consequentially, alterations in life‐history strategies are closely intertwined with such interaction processes. Understanding the connection between life‐history alterations and host–parasite interactions can therefore be important to unveil potential links between adaptation to environmental change and changes in interaction processes. Here, we studied how two different host–parasite interaction processes, oral and hemocoelic exposure to bacteria, affect various life histories of the Glanville fritillary butterfly Melitaea cinxia. We either fed or injected adult butterflies with the bacterium Micrococcus luteus and observed for differences in immune defenses, reproductive life histories, and longevity, compared to control exposures. Our results indicate differences in how female butterflies adapt to the two exposure types. Orally infected females showed a reduction in clutch size and an earlier onset of reproduction, whereas a reduction in egg weight was observed for hemocoelically exposed females. Both exposure types also led to shorter intervals between clutches and a reduced life span. These results indicate a relationship between host–parasite interactions and changes in life‐history strategies. This relationship could cast restrictions on the ability to adapt to new environments and consequentially influence the population dynamics of a species in changing environmental conditions., Here, we report differences in how female butterflies adapt life‐history traits upon two distinct infection modes. Oral exposure resulted in a accelerated decrease of clutch sizes but an increase in clutch number, whereas hemocoelic exposure resulted in a reduction of egg weight.

Bacterial infection commonly complicates inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). The mechanisms of increased infection susceptibility and how use of the commonly prescribed therapy inhaled corticosteroids (ICS) accentuates pneumonia risk in COPD are poorly understood. Here, using analysis of samples from patients with COPD, we show that ICS use is associated with lung microbiota disruption leading to proliferation of streptococcal genera, an effect that could be recapitulated in ICS-treated mice. To study mechanisms underlying this effect, we used cellular and mouse models of streptococcal expansion with Streptococcus pneumoniae, an important pathogen in COPD, to demonstrate that ICS impairs pulmonary clearance of bacteria through suppression of the antimicrobial peptide cathelicidin. ICS impairment of pulmonary immunity was dependent on suppression of cathelicidin because ICS had no effect on bacterial loads in mice lacking cathelicidin (Camp−/−) and exogenous cathelicidin prevented ICS-mediated expansion of streptococci within the microbiota and improved bacterial clearance. Suppression of pulmonary immunity by ICS was mediated by augmentation of the protease cathepsin D. Collectively, these data suggest a central role for cathepsin D/cathelicidin in the suppression of antibacterial host defense by ICS in COPD. Therapeutic restoration of cathelicidin to boost antibacterial immunity and beneficially modulate the lung microbiota might be an effective strategy in COPD.

Background Neisseriameningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD) but the contribution of rare variants other than complement has not been determined. Methods We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-threatening Infectious Disease Study. Candidate genetic variants were identified by whole exome sequencing of two patients with familial IMD. Candidate variants were further validated by in vitro assays. Results Exomes of two siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other non-familial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defence protein expressed in the nasopharyngeal epithelia, however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant rSPLUNC1 (p.G22E) showed reduced anti-biofilm activity, increased meningococcal adhesion and invasion of cells compared with wild type SPLUNC1. Conclusions A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

Hyperoxia-induced injury to the developing lung, impaired alveolarization, and dysregulated vascularization are critical factors in the pathogenesis of bronchopulmonary dysplasia (BPD); however, mechanisms for hyperoxia-induced development of BPD are not fully known. In this study, we show that TREM-1 (triggering receptor expressed on myeloid cells 1) is upregulated in hyperoxia-exposed neonatal murine lungs as well as in tracheal aspirates and lungs of human neonates with respiratory distress syndrome and BPD as an adaptive response to survival in hyperoxia. Inhibition of TREM-1 function using an siRNA approach or deletion of the Trem 1 gene in mice showed enhanced lung inflammation, alveolar damage, and mortality of hyperoxia-exposed neonatal mice. The treatment of hyperoxia-exposed neonatal mice with agonistic TREM-1 antibody decreased lung inflammation, improved alveolarization, and was associated with diminished necroptosis-regulating protein RIPK3 (receptor-interacting protein kinase 3). Mechanistically, we show that TREM-1 activation alleviates lung inflammation and improves alveolarization through downregulating RIPK3-mediated necroptosis and NLRP3 (nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3) inflammasome activation in hyperoxia-exposed neonatal mice. These data show that activating TREM-1, enhancing angiopoietin 1 signaling, or blocking the RIPK3-mediated necroptosis pathway may be used in new therapeutic interventions to control adverse effects of hyperoxia in the development of BPD.