Luminal bacteria coated with IgA and IgG during intestinal inflammation as a new and abundant stimulus for colonic macrophages.

In the gut, secretory immunoglobulin A is the predominant humoral response against commensals, although healthy hosts also produce microbiota-specific IgG antibodies. During intestinal inflammation, the content of IgG in the lumen increases along with the proportion of commensal bacteria coated with this antibody, suggesting signalling through the IgG-CD64 axis in the pathogenesis of inflammatory bowel diseases. In this work, we evaluated day by day the frequency of faecal bacteria coated with IgA and IgG during the development of DSS colitis. We studied the phenotype and phagocytic activity of F4/80+ CD64+ colonic macrophages, as well as the production of cytokines and nitric oxide by lamina propria or bone marrow-derived macrophages after stimulation with IgA+ , IgG+ and IgA+ IgG+ bacteria. We found that the percentage of faecal IgA+ IgG+ double-coated bacteria increased rapidly during DSS colitis. Also, analysis of the luminal content of mice with colitis showed a markedly superior ability to coat fresh bacteria. IgA+ IgG+ bacteria were the most potent stimulus for phagocytic activity involving CD64 and Dectin-1 receptors. IgA+ IgG+ bacteria observed during the development of DSS colitis could represent a new marker to monitor permeability and inflammatory progression. The interaction of IgA+ IgG+ bacteria with CD64+ F4/80+ macrophages could be part of the complex cascade of events in colitis. Interestingly, after stimulation, CD64+ colonic macrophages showed features similar to those of restorative macrophages that are relevant for tissue repair and healing.

γδ T cells exhibit multifunctional and protective memory in intestinal tissues.

The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αß T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa. Challenge infection with oral Lm, but not oral Salmonella or intravenous Lm, induced rapid expansion of memory γδ T cells, suggesting contextual specificity to the priming pathogen. Importantly, memory γδ T cells were able to provide enhanced protection against infection. These findings illustrate that γδ T cells play a role with hallmarks of adaptive immunity in the intestinal mucosa.

IL-17A-producing resident memory γδ T cells orchestrate the innate immune response to secondary oral Listeria monocytogenes infection.

Memory γδ T cells are important for the clearance of Listeria monocytogenes infection in the intestinal mucosa. However, the mechanisms by which memory γδ T cells provide protection against secondary oral infection are poorly understood. Here we used a recombinant strain of L. monocytogenes that efficiently invades the intestinal epithelium to show that Vγ4(+) memory γδ T cells represent a resident memory (Trm) population in the mesenteric lymph nodes (MLNs). The γδ Trm exhibited a remarkably static pattern of migration that radically changed following secondary oral L. monocytogenes infection. The γδ Trms produced IL-17A early after rechallenge and formed organized clusters with myeloid cells surrounding L. monocytogenes replication foci only after a secondary oral infection. Antibody blocking studies showed that in addition to IL-17A, the chemokine receptor C-X-C chemokine receptor 3 (CXCR3) is also important to enable the local redistribution of γδ Trm cells and myeloid cells specifically near the sites of L. monocytogenes replication within the MLN to restrict bacterial growth and spread. Our findings support a role for γδ Trms in orchestrating protective immune responses against intestinal pathogens.

Platelets support a protective immune response to LCMV by preventing splenic necrosis.

Severe arenaviral infections in humans are characterized by clinical findings common to other viral hemorrhagic fevers (VHFs), including thrombocytopenia, leukopenia, skin and internal organ hemorrhages, high viral replication, splenic necrosis, and death. Host responses, rather than direct damage by the arenaviral replication, account for most of the observed pathology, but it is not known what protective roles platelets may have in each of the manifestations. To address this issue in an animal model, we compared nondepleted (100%), partially depleted (15%), and profoundly (< 2.5%) platelet depleted mice infected with the mouse arenavirus lymphocytic choriomeningitis virus (LCMV). Here, we describe that systemic bleedings and death were seen only in those animals receiving the stronger depletion treatment. Furthermore, we showed that the nonhemorrhagic but partially platelet-depleted mice were unable to control the viral replication because of generalized splenic necrosis, affecting innate and adaptive immune cells.These data suggest that, by their supportive roles in hemostasis, platelets may be preventing the severe pathology observed in human arenaviral infections.

Transient FTY720 treatment promotes immune-mediated clearance of a chronic viral infection.

For a wide variety of microbial pathogens, the outcome of the infection is indeterminate. In some individuals the microbe is cleared, but in others it establishes a chronic infection, and the factors that tip this balance are often unknown. In a widely used model of chronic viral infection, C57BL/6 mice clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), but the clone 13 strain persists. Here we show that the Armstrong strain induces a profound lymphopenia at days 1-3 after infection, but the clone 13 strain does not. If we transiently augment lymphopenia by treating the clone-13-infected mice with the drug FTY720 at days 0-2 after infection, the mice successfully clear the infection by day 30. Clearance does not occur when CD4 T cells are absent at the time of treatment, indicating that the drug is not exerting direct antiviral effects. Notably, FTY720 treatment of an already established persistent infection also leads to viral clearance. In both models, FTY720 treatment preserves or augments LCMV-specific CD4 and CD8 T-cell responses, a result that is counter-intuitive because FTY720 is generally regarded as a new immunosuppressive agent. Because FTY720 targets host pathways that are completely evolutionarily conserved, our results may be translatable into new immunotherapies for the treatment of chronic microbial infections in humans.

New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

Serum Biomarkers for the Prediction of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of global cancer death. Major etiologies of HCC relate to chronic viral infections as well as metabolic conditions. The survival rate of people with HCC is very low and has been attributed to late diagnosis with limited treatment options. Combining ultrasound and the biomarker alpha-fetoprotein (AFP) is currently one of the most widely used screening combinations for HCC. However, the clinical utility of AFP is controversial, and the frequency and operator-dependence of ultrasound lead to a variable degree of sensitivity and specificity across the globe. In this review, we summarize recent developments in the search for non-invasive serum biomarkers for early detection of HCC to improve prognosis and outcome for patients. We focus on tumor-associated protein markers, immune mediators (cytokines and chemokines), and micro-RNAs in serum or circulating extracellular vesicles and examine their potential for clinical application.

Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection.

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlAMLm). InlAMLm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAMLm initially disseminated from the gut to the MLN normally in Batf3-/- mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAMLm. At this time Batf3-/- mice displayed reduced InlAMLm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3-/- mice also exhibited profound defects in the induction and gut-homing of InlAMLm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3-/- mice, indicating a critical role for Batf3 in generating anti-InlAMLm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAMLm infection and in driving the establishment of intestinal Lm-specific effector T cells.

Association between cardiovascular disease risk scores and subclinical atherosclerosis prevalence in non-elderly adult patients from Argentina.

The goal of our study was to use statistical analysis to try to associate cardiovascular disease (CVD) risk scores and the observed prevalence of subclinical atherosclerosis (SA) in a non-elderly adult local population. An observational cross-sectional study was carried out (143 male and 131 female) on non-elderly adults (20-59 years). CVD risk scores included Framingham Risk Scores for 10-year hard (FRS 10 H), 30-year lipid hard or CVD (FRS 30 L H or FRS 30 L CVD), 30 year-body mass index hard or CVD (FRS 30 BMI H or FRS 30 BMI CVD) and Pooled Cohort Risk Equations for either 10 years (PCE 10) or lifetime (PCE LT). The Carotid Ultrasound (CU) study was performed and the Coronary Artery Calcium (CAC) score were obtained to assess SA. The Receiving Operating Characteristic (ROC) curve analysis followed by Youden's index was used to evaluate and adjust the stratification of CVD risk scores. SA was detected in 32.4% of individuals. The risk scores that showed the biggest areas under the ROC curve were FRS 30 L (H and CVD). When the cut-off values for these CVD risk scores were adjusted, the FRS 30 L H increased the negative predictive value for the low risk group from 87.7 to 97.0% and the FRS 30 L CVD increased the positive predictive values for the high risk group from 69.7 to 85.7%. The CVD risk stratification of non-elderly adults using FRS 30 L H and FRS 30 L CVD may be a useful tool for selecting candidate patients for diagnostic imaging studies that assess their SA prevalence.

CD169+ macrophages orchestrate innate immune responses by regulating bacterial localization in the spleen.

The spleen is an important site for generating protective immune responses against pathogens. After infection, immune cells undergo rapid reorganization to initiate and maintain localized inflammatory responses; however, the mechanisms governing this spatial and temporal cellular reorganization remain unclear. We show that the strategic position of splenic marginal zone CD169+ macrophages is vital for rapid initiation of antibacterial responses. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrate a second phase of innate protection by mediating the transport of bacteria to splenic T cell zones. This compartmentalization of bacteria within the spleen was essential for driving the reorganization of innate immune cells into hierarchical clusters and for local interferon-γ production near sites of bacterial replication foci. Our results show that both phases of the antimicrobial innate immune response were dependent on CD169+ macrophages, and, in their absence, the series of events needed for pathogen clearance and subsequent survival of the host was disrupted. Our study provides insight into how lymphoid organ structure and function are related at a fundamental level.

CD8 T cell memory recall is enhanced by novel direct interactions with CD4 T cells enabled by MHC class II transferred from APCs.

Protection against many intracellular pathogens is provided by CD8 T cells, which are thought to need CD4 T cell help to develop into effective memory CD8 T cells. Because murine CD8 T cells do not transcribe MHC class II (MHC-II) genes, several models have proposed antigen presenting cells (APCs) as intermediaries required for CD4 T cells to deliver their help to CD8 T cells. Here, we demonstrate the presence of MHC-II molecules on activated murine CD8 T cells in vitro as well as in vivo. These MHC-II molecules are acquired via trogocytosis by CD8 T cells from their activating APCs, particularly CD11c positive dendritic cells (DCs). Transferred MHC-II molecules on activated murine CD8 T cells were functionally competent in stimulating specific indicator CD4 T cells. CD8 T cells that were "helped" in vitro and subsequently allowed to rest in vivo showed enhanced recall responses upon challenge compared to "helpless" CD8 T cells; in contrast, no differences were seen upon immediate challenge. These data indicate that direct CD8:CD4 T cell interactions may significantly contribute to help for CD8 T cells. Furthermore, this mechanism may enable CD8 T cells to communicate with different subsets of interacting CD4 T cells that could modulate immune responses.

Cryptosporidium parvum: effect of multi-drug reversing agents on the expression and function of ATP-binding cassette transporters.

In the present study, the gene expression of three multidrug resistance (MDR) and resistance-associated protein (MRP) transport proteins or efflux pumps was characterized and the phenotypic evidence for such pumps was demonstrated in cultured Madin-Darby canine kidney (MDCK) cells. A gradient for the fluorescent probe calcein was established between parasite and host cell suggestive of a parasite extrusion pump at the parasite-host interface. This gradient was decreased in a glucose-free medium containing 2-deoxyglucose or 3-O-methylglucose, by probenecid, and by the isoflavonoid, narigenin, suggesting that the calcein extrusion was energy-dependent and involved an MRP-like pump. While neither MDR or MRP inhibiters significantly affected transcript levels of any of the ABC transporters, transcript levels of the Cryptosporidium parvum ABC protein (CpABC1), an MRP transporter, were consistently expressed 4 logs higher than either CpABC3 or CpABC2, suggesting a prominent role in the intracellular stages of the parasite.