Early Time Points Of Infection Research Articles

Context-dependent role of group 3 innate lymphoid cells in mucosal protection.

How group 3 innate lymphoid cells (ILC3s) regulate mucosal protection in the presence of T cells remains poorly understood. Here, we examined ILC3 function in intestinal immunity using ILC3-deficient mice that maintain endogenous T cells, T helper 17 (TH17) cells, and secondary lymphoid organs. ILC3s were dispensable for generation of TH17 and TH22 cell responses to commensal and pathogenic bacteria, and absence of ILC3s did not affect IL-22 production by CD4 T cells before or during infection. However, despite the presence of IL-22-producing T cells, ILC3s and ILC3-derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. T cell-sufficient, ILC3-deficient mice were capable of pathogen clearance and survived infection with a low dose of Citrobacter rodentium. However, ILC3s promoted pathogen tolerance at early time points of infection by activating tissue-protective immune pathways. Consequently, ILC3s were indispensable for survival after high-dose infection. Our results demonstrate a context-dependent role for ILC3s in immune-sufficient animals and provide a blueprint for uncoupling of ILC3 and TH17 cell functions.

Multi-omic profiling reveals early immunological indicators for identifying COVID-19 Progressors

We sought to better understand the immune response during the immediate post-diagnosis phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14−CD16+ and intermediate CD14+CD16+ monocytes. In lymphocytes, the CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. These early stage observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19. BackgroundMuch of the literature on immune response post-SARS-CoV-2 infection has been in the acute and post-acute phases of infection. Translational significanceWe found differences at early time points of infection in approximately 160 participants. We compared multi-omic signatures in immune cells between individuals progressing to needing more significant medical intervention and non-progressors. We observed widespread evidence of a state of increased inflammation associated with progression, supported by a range of epigenomic, transcriptomic, and proteomic signatures. The signatures we identified support other findings at later time points and serve as the basis for prognostic biomarker development or to inform interventional strategies.

Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions

In this work, we investigated the presence and function of TRPM8, a non-selective and cold-sensitive Ca2+-permeable ion channel in the primary microglia cell as well as in microglia cell line BV2. We demonstrate that primary microglia as well as BV2 express TRPM8 endogenously. Both pharmacological activation or inhibition of TRPM8 causes enhanced uptake of bacterial particles at early time points of infection. In BV2, TRPM8 activation and/or LPS-signaling alters its surface expression and cytosolic ROS production. TRPM8 modulation in the absence and presence of LPS causes differential regulation of cytosolic pH and lysosomal pH. Notably, TRPM8 modulation also alters the correlation between lysosomal pH and cytosolic pH depending on TRPM8 modulation and the presence or absence of LPS. Collectively our data suggest that TRPM8 is involved in the regulation of subcellular organelle, i.e. mitochondrial and lysosomal functions. Data also suggest that primarily TRPM8 activation, but often deviation from endogenous TRPM8 function is linked with better innate immune function mediated by microglial cells. We suggest that TRPM8-mediated regulations of sub-cellular organelle functions are more context-dependent manner. Such understanding is relevant in the context of microglial cell functions and innate immunity.

Comparative microRNA profiling of Trypanosoma cruzi infected human cells.

Trypanosoma cruzi, the causative agent of Chagas disease, can infect almost any nucleated cell in the mammalian host. Although previous studies have described the transcriptomic changes that occur in host cells during parasite infection, the understanding of the role of post-transcriptional regulation in this process is limited. MicroRNAs, a class of short non-coding RNAs, are key players in regulating gene expression at the post-transcriptional level, and their involvement in the host-T. cruzi interplay is a growing area of research. However, to our knowledge, there are no comparative studies on the microRNA changes that occur in different cell types in response to T. cruzi infection. Here we investigated microRNA changes in epithelial cells, cardiomyocytes and macrophages infected with T. cruzi for 24 hours, using small RNA sequencing followed by careful bioinformatics analysis. We show that, although microRNAs are highly cell type-specific, a signature of three microRNAs -miR-146a, miR-708 and miR-1246, emerges as consistently responsive to T. cruzi infection across representative human cell types. T. cruzi lacks canonical microRNA-induced silencing mechanisms and we confirm that it does not produce any small RNA that mimics known host microRNAs. We found that macrophages show a broad response to parasite infection, while microRNA changes in epithelial and cardiomyocytes are modest. Complementary data indicated that cardiomyocyte response may be greater at early time points of infection. Our findings emphasize the significance of considering microRNA changes at the cellular level and complement previous studies conducted at higher organizational levels, such as heart samples. While miR-146a has been previously implicated in T. cruzi infection, similarly to its involvement in many other immunological responses, miR-1246 and miR-708 are demonstrated here for the first time. Given their expression in multiple cell types, we anticipate our work as a starting point for future investigations into their role in the post-transcriptional regulation of T. cruzi infected cells and their potential as biomarkers for Chagas disease.

Induction of MMP-3 and MMP-9 expression during Helicobacter pylori infection via MAPK signaling pathways.

Helicobacter pylori (H. pylori)-induced gastric pathology involves remodeling of extracellular matrix mediated by aberrant activity of matrix metalloproteinases (MMPs). We have previously shown that in vitro H. pylori infection leads to MMP-3 and MMP-9 overexpression, associated with phosphorylation of bacterial oncoprotein CagA. We extended these findings in an in vivo model of H. pylori infection and further assessed the involvement of MAPK pathways in MMP expression. C57BL/6 mice were infected with H. pylori strains HPARE, HPARE ΔCagA, and SS1, for 6 and 9 months. Transcriptional expression of Mmp-3 and Mmp-9 was evaluated via qPCR while respective protein levels in the gastric mucosa were determined immunohistochemically. Epithelial cell lines AGS and GES-1 were infected with H. pylori strain P12 in the presence of chemical inhibitors of JNK, ERK1/2, and p38 pathways, for 24 h. mRNA and protein expression of MMP-3 and MMP-9 were determined via qPCR and Western blot, respectively. We observed transcriptional activation of Mmp-3 and Mmp-9 as well as aberrant MMP-3 and MMP-9 protein expression in murine gastric tissue following H. pylori infection. CagA expression was associated with MMP upregulation, particularly during the early time points of infection. We found that inhibition of ERK1/2 resulted in reduced mRNA and protein expression of MMP-3 and MMP-9 during H. pylori infection, in both cell lines. Expressed protein levels of both MMPs were also found reduced in the presence of JNK pathway inhibitors in both cell lines. However, p38 inhibition resulted in a more complex effect, probably attributed to the accumulation of phospho-p38 and increased phospho-ERK1/2 activity due to crosstalk between MAPK pathways. H. pylori colonization leads to the upregulation of MMP-3 and MMP-9 in vivo, which primarily involves ERK1/2 and JNK pathways. Therefore, their inhibition may potentially offer a protective effect against gastric carcinogenesis and metastasis.

A24 INVESTIGATING THE IMPACT OF PARKINSON’S DISEASE-ASSOCIATED GENES ON INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells (IECs) provide an essential physical barrier between harsh luminal contents and underlying host tissue. The maintenance of intestinal homeostasis in this rapidly renewing tissue must be intricately regulated through the proliferation and differentiation of intestinal stem cells (ISCs). Dysregulation of this system results in the loss of barrier function, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, there is increasing evidence linking PD progression and gastrointestinal dysfunction. For instance, constipation and increased bowel permeability are frequently observed years prior to development of motor dysfunction in PD, people with inflammatory bowel disease are more likely to develop PD, and a positive correlation exists between gastrointestinal infections and PD incidence. Our group recently developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with Gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 and other PD-associated genes are expressed in IECs, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology. Purpose Investigate the impact of Pink1 and other PD-associated genes in IECs under steady state and infection. Method Single-cell RNA sequencing was performed on IECs isolated from Pink1 WT and KO mice, at steady state and following in vivo C. rodentium infection. Mice were sacrificed at an early timepoint of infection (day 6) to elucidate transcriptional differences between epithelial lineages of each genotype. Additionally, ex vivo colonoids were derived from primary mouse tissue and treated with lipopolysaccharide (LPS) to determine how PINK1 loss-of-function affects the inflammatory response of the epithelium. Result(s) Our data revealed that loss-of-function of PINK1 profoundly affected the ISC compartment and several epithelial lineages. Specifically, ISCs from infected Pink1 KO mice demonstrated differentially regulated proliferative and cell cycle genes, while transit amplifying cells showed dysregulated expression of tight junction genes, and enterocytes displayed differentially expressed oxidative damage and apoptotic genes. Preliminary data from colonoids showed that Pink1 KO mice, when stimulated with LPS, had increased pro-inflammatory cytokine gene expression. Conclusion(s) In Pink1 KO intestinal epithelial cells, there is indeed an altered cellular response upon infection in vivo and LPS treatment ex vivo. However, more information is needed to decern the mechanistic role of IECs in PD. By investigating the role of PD genes in the gastrointestinal tract, these studies carry important implications for understanding the initiation and progression of PD. Disclosure of Interest None Declared

Novel approaches for the serodiagnosis of louse-borne relapsing fever.

Louse-borne relapsing fever (LBRF) caused by B. recurrentis is a poverty-related and neglected infectious disease with an endemic focus in the Horn of Africa. Re-emergence of the disease occurred in Europe during the refugee crisis in 2015 and sporadic outbreaks were frequently reported in Eastern Africa where poor settings lack affordable diagnostics. Currently, there are no validated in vitro assays available for the serodiagnosis of LBRF. The aim of this study was to develop novel and reliable immunoassays by investigating clinically suspected and culture-confirmed serum samples from LBRF patients and a broad panel of serum samples from patients with other spirochetal, bacterial, and parasitic diseases. We identified two immunoreactive antigens (complement-inhibiting protein CihC and the glycerophosphodiester phosphodiesterase GlpQ of B. recurrentis) as the most promising target candidates leading to the evaluation of two immunoassays (line immunoblot and ELISA) for IgM and IgG. To optimize the IgM immunoassay, we conducted a bioinformatic approach to localize the relevant immunogenic regions within CihC. By utilizing a N-terminal CihC fragment, the sensitivity and specificity of both immunoassays (CihC and GlpQ) were high (IgM: sensitivity 100%, specificity of 89.9%, IgG: sensitivity 100%, specificity 99.2%). In conclusion, our findings indicate the diagnostic potential of CihC and GlpQ as valuable markers for the serodiagnosis of LBRF even at early time points of infection. Here, we provide strong evidence for the utilization of these immunoassays as reliable tools in clinical practice.

Multi-tissue RNAseq reveals genetic and temporal differences in acute response to viral (IHNV) infection among three selected lines of rainbow trout with varying resistance

Utilizing RNA-seq, this study compared the transcriptomic responses of three improved strains (VSel, PSel, and CSel) of rainbow trout fry during acute stages of challenge with infectious hematopoietic necrosis virus (IHNV). The VSel strain has been selected for resistance against the specific strain of IHNV used in our challenge, PSel has undergone selection for utilization of plant-protein based feeds and previously has shown elevated non-specific disease resistance despite no disease related selection pressures, and the final strain, CSel, is a commercial strain that has been domesticated for several years but has not been selected for specific viral disease resistance. Following a 21-day IHNV challenge, Kaplan-Meier survival estimator curves and cumulative percent mortality (CPM) showed significant differences in IHNV resistance across strains: VSel - 19.3 ± 5.0%, PSel - 67. ± 3.03%, CSel - 94.6 ± 4.1% CPM. To evaluate acute responses to IHNV infection, whole blood, as well as samples from the kidney, liver, and intestine, were collected at 0, 4, 12, 24, and 48 h post infection (hpi). Serum lysozyme activity, a marker of non-specific innate immunity, showed strain and temporal effects during the acute infection phase with PSel showing the highest activity at 0 and 48 hpi. Differential gene expression responses were detected, with varying degrees, in all tissues, both between strains, as well as across acute timepoints within strains. The VSel strain showed upregulation for a particular subset of viral recognition genes during early infection timepoints and rather limited upregulation of immune genes later, while maintaining and reactivating metabolic pathways. The CSel strain showed a downregulation of metabolic related genes and a limited upregulation of immune genes, while the PSel strain showed similar downregulation of metabolic genes during acute infection, yet when compared to the CSel strain, showed a more robust innate immune response. Evaluation of upregulated immune response genes, as well as interferon-related genes showed the PSel strain to have the greatest number of uniquely upregulated immune genes in both the kidney and intestine, with CSel and PSel showing a similar number of such genes upregulated in liver. A moderate number of immune response genes were shared between PSel and CSel in all tissues, though both PSel and VSel showed a high number of uniquely overexpressed immune response genes in the kidney, and PSel showed the highest number of uniquely upregulated interferon related genes in the intestine. Overall, the VSel response was unique from the CSel with very little overlap in activated immune responses. Findings from this study highlight the disparity in IHNV resistance among genetic strains of rainbow trout, while identifying molecular mechanisms underlying differences in disease phenotypes. Furthermore, our results on trout strains with distinct selection backgrounds yields comparative insights into the adaptive gains brought about by selection programs for pathogen-specific disease resistance, as well as the non-specific immune enhancement associated with selection for utilization of plant-based diets.

Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions.

Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.

Gene Expression Response to Stony Coral Tissue Loss Disease Transmission in M. cavernosa and O. faveolata From Florida

Since 2014, corals within Florida’s Coral Reef have been dying at an unprecedented rate due to stony coral tissue loss disease (SCTLD). Here we describe the transcriptomic outcomes of three different SCTLD transmission experiments performed at the Smithsonian Marine Station and Mote Marine Laboratory between 2019 and 2020 on the corals Orbicella faveolata and Montastraea cavernosa. Overall, diseased O. faveolata had 2194 differentially expressed genes (DEGs) compared with healthy colonies, whereas diseased M. cavernosa had 582 DEGs compared with healthy colonies. Many significant DEGs were implicated in immunity, extracellular matrix rearrangement, and apoptosis. These included, but not limited to, peroxidases, collagens, Bax-like, fibrinogen-like, protein tyrosine kinase, and transforming growth factor beta. A gene module was identified that was significantly correlated to disease transmission. This module possessed many apoptosis and immune genes with high module membership indicating that a complex apoptosis and immune response is occurring in corals during SCTLD transmission. Overall, we found that O. faveolata and M. cavernosa exhibit an immune, apoptosis, and tissue rearrangement response to SCTLD. We propose that future studies should focus on examining early time points of infection, before the presence of lesions, to understand the activating mechanisms involved in SCTLD.

Primary HIV-1 and Infectious Molecular Clones Are Differentially Susceptible to Broadly Neutralizing Antibodies.

To prevent the spread of HIV-1, a vaccine should elicit antibodies that block viral entry for all cell types. Recently, we have developed a virus capture assay to quantitatively examine early time points of infection. Here we present data on the ability of bNAbs to inhibit capture (1 h) or replication (48 h) of purified primary acute or chronic HIV or infectious molecular clones (IMCs) in human peripheral blood mononuclear cells (PBMCs) as quantified by qRT-PCR. Although bNAbs significantly inhibited HIV-1 replication in PBMCs in a virus subtype and in a PBMC-donor specific manner, they did not inhibit virus capture of primary viruses. In contrast, IMC capture and replication in PBMCs and purified CD4+ T cells were significantly inhibited by bNAbs, thus indicating that unlike IMCs, primary HIV-1 may initially bind to other cell surface molecules, which leads to virus capture even in the presence of bNAbs. Our results demonstrate that the initial interactions and some aspects of infectivity of primary HIV-1 and IMCs are inherently different, which underscores the importance of studying virus transmission using primary viruses in in vitro studies, an issue that could impact HIV-1 vaccine design strategies.

Control of Persistent Salmonella Infection Relies on Constant Thymic Output Despite Increased Peripheral Antigen-Specific T Cell Immunity.

Recent thymic emigrants are the youngest subset of peripheral T cells and their involvement in combating persistent bacterial infections has not been explored. Here, we hypothesized that CD4+ recent thymic emigrants are essential immune mediators during persistent Salmonella infection. To test this, we thymectomized adult mice either prior to, or during, persistent Salmonella infection. We found that thymic output is crucial in the formation of protective immune responses during the early formation of a Salmonella infection but is dispensable once persistent Salmonella infection is established. Further, we show that thymectomized mice demonstrate increased infection-associated mortality and bacterial burdens. Unexpectedly, numbers of Salmonella-specific CD4+ T cells were significantly increased in thymectomized mice compared to sham control mice. Lastly, we found that T cells from thymectomized mice may be impaired in producing the effector cytokine IL-17 at early time points of infection, compared to thymically intact mice. Together, these results imply a unique role for thymic output in the formation of immune responses against a persistent, enteric pathogen.

Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress.

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

Cooperative functioning of salicylic acid and phenylalanine ammonia lyase in barley plant challenged with spot blotch and powdery mildew diseases

Salicylic acid (SA) and phenylalanine ammonia-lyase (PAL) have been suggested as important signals during plant resistance towards several fungal pathogens. In this work, to better understand the defense responses initiated by resistant and susceptible barley genotypes challenged with a necrotrophic (Cochliobolus sativus; Cs) and a biotrophic (Blumeria graminis; Bg) pathogens, the relative contributions of SA and PAL were investigated at early time points of infection. SA signaling was activated in both genotypes 24 hours post infection (hpi) as compared with the non-inoculated plants. However, with or without pathogen pretreatment, SA significantly increased (P = 0.001) in the resistant genotype that contained three-folds of total SA in comparison with the susceptible one for Bg. Reverse transcription-polymerase chain reaction (RTPCR) analysis revealed that PAL expression increases in the resistant and susceptible genotypes over the inoculation time points, with the maximum expression observed 48 hpi. PAL expression was paralleled by an increase in SA content in leaves as shown by the test coincidence (F3, 32 = 1.09, P = 0.49 for Cs and F3, 32 = 1.03, P = 0.48 for Bg). Results showed that the cooperatively function of SA and PAL in barley responses to both Cs and Bg appeared to be dependent on the plant genotype, and that SA signaling and PAL play a role in barley interactions with these both pathogens. This study might increase our understanding for a deeper molecular research on barley defense responses against pathogens with different lifestyles.

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bacteria. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated at early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.

Rapid loss of early antigen-presenting activity of lymph node dendritic cells against Ag85A protein following Mycobacterium bovis BCG infection

BackgroundControl of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood.ResultsIn the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette–Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo.ConclusionsOur results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

Infection of a tomato cell culture by Phytophthora infestans; a versatile tool to study Phytophthora-host interactions

BackgroundThe oomycete Phytophthora infestans causes late blight on potato and tomato. Despite extensive research, the P. infestans-host interaction is still poorly understood. To find new ways to further unravel this interaction we established a new infection system using MsK8 tomato cells. These cells grow in suspension and can be maintained as a stable cell line that is representative for tomato.ResultsMsK8 cells can host several Phytophthora species pathogenic on tomato. Species not pathogenic on tomato could not infect. Microscopy revealed that 16 h after inoculation up to 36% of the cells were infected. The majority were penetrated by a germ tube emerging from a cyst (i.e. primary infection) while other cells were already showing secondary infections including haustoria. In incompatible interactions, MsK8 cells showed defense responses, namely reactive oxygen species production and cell death leading to a halt in pathogen spread at the single cell level. In compatible interactions, several P. infestans genes, including RXLR effector genes, were expressed and in both, compatible and incompatible interactions tomato genes involved in defense were differentially expressed.ConclusionsOur results show that P. infestans can prosper as a pathogen in MsK8 cells; it not only infects, but also makes haustoria and sporulates, and it receives signals that activate gene expression. Moreover, MsK8 cells have the ability to support pathogen growth but also to defend themselves against infection in a similar way as whole plants. An advantage of MsK8 cells compared to leaves is the more synchronized infection, as all cells have an equal chance of being infected. Moreover, analyses and sampling of infected tissue can be performed in a non-destructive manner from early time points of infection onwards and as such the MsK8 infection system offers a potential platform for large-scale omics studies and activity screenings of inhibitory compounds.

Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis.

To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity.

IL-22 dampens the T cell response in experimental malaria.

A tight regulation between the pro– and anti–inflammatory immune responses during plasmodial infection is of crucial importance, since a disruption leads to severe malaria pathology. IL-22 is a member of the IL-10 cytokine family, which is known to be highly important in immune regulation. We could detect high plasma levels of IL-22 in Plasmodium falciparum malaria as well as in Plasmodium berghei ANKA (PbA)-infected C57BL/6J mice. The deficiency of IL-22 in mice during PbA infection led to an earlier occurrence of cerebral malaria but is associated with a lower parasitemia compared to wt mice. Furthermore, at an early time point of infection T cells from PbA-infected Il22−/− mice showed an enhanced IFNγ but a diminished IL-17 production. Moreover, dendritic cells from Il22−/− mice expressed a higher amount of the costimulatory ligand CD86 upon infection. This finding can be corroborated in vitro since bone marrow-derived dendritic cells from Il22−/− mice are better inducers of an antigen-specific IFNγ response by CD8+ T cells. Even though there is no IL-22 receptor complex known on hematopoietic cells, our data suggest a link between IL-22 and the adaptive immune system which is currently not identified.