GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells.

Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4(+) T cells undergo less-efficient homeostatic proliferation than CD8(+) T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-alpha-positive (IL-7Ralpha(+)) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4(+) T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Ralpha(+) DCs. Our results indicate that IL-7Ralpha(+) DCs are regulators of the peripheral CD4(+) T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4(+) T cell population expansion in vivo.

Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly.

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.

Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks.

OBJECTIVE: Despite widespread use of antibiotics for the treatment of life-threatening infections and for research on the role of commensal microbiota, our understanding of their effects on the host is still very limited. DESIGN: Using a popular mouse model of microbiota depletion by a cocktail of antibiotics, we analysed the effects of antibiotics by combining intestinal transcriptome together with metagenomic analysis of the gut microbiota. In order to identify specific microbes and microbial genes that influence the host phenotype in antibiotic-treated mice, we developed and applied analysis of the transkingdom network. RESULTS: We found that most antibiotic-induced alterations in the gut can be explained by three factors: depletion of the microbiota; direct effects of antibiotics on host tissues and the effects of remaining antibiotic-resistant microbes. Normal microbiota depletion mostly led to downregulation of different aspects of immunity. The two other factors (antibiotic direct effects on host tissues and antibiotic-resistant microbes) primarily inhibited mitochondrial gene expression and amounts of active mitochondria, increasing epithelial cell death. By reconstructing and analysing the transkingdom network, we discovered that these toxic effects were mediated by virulence/quorum sensing in antibiotic-resistant bacteria, a finding further validated using in vitro experiments. CONCLUSIONS: In addition to revealing mechanisms of antibiotic-induced alterations, this study also describes a new bioinformatics approach that predicts microbial components that regulate host functions and establishes a comprehensive resource on what, why and how antibiotics affect the gut in a widely used mouse model of microbiota depletion by antibiotics.

Lipopolysaccharide-activated dendritic cells: "exhausted" or alert and waiting?

LPS-activated dendritic cells (DCs) are thought to follow a set program in which they secrete inflammatory cytokines (such as IL-12) and then become refractory to further stimulation (i.e., "exhausted"). In this study, we show that mouse DCs do indeed lose their responsiveness to LPS, but nevertheless remain perfectly capable of making inflammatory cytokines in response to signals from activated T cells and to CD40-ligand and soluble T cell-derived signals. Furthermore, far from being rigidly programmed by the original activating stimulus, the DCs retained sufficient plasticity to respond differentially to interactions with Th0, Th1, Th2, and Th17 T cells. These data suggest that LPS activation does not exhaust DCs but rather primes them for subsequent signals from T cells.

Resident peritoneal NK cells.

In this study, we describe a new population of NK cells that reside in the normal, uninflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b(-), CD27(+) immature splenic NK cells, as well as liver NK cells, but they differ in their expression of CD62L, TRAIL, and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF, and TNF-α and in the killing of YAC-1 target cells. We also found that the peritoneum induces different behavior in mature and immature splenic NK cells. When transferred i.v. into RAGγc knockout mice, both populations undergo homeostatic proliferation in the spleen, but only the immature splenic NK cells are able to reach the peritoneum. When transferred directly into the peritoneum, the mature NK cells survive but do not divide, whereas the immature NK cells proliferate profusely. These data suggest that the peritoneum is not only home to a new subset of tissue-resident NK cells, but that it differentially regulates the migration and homeostatic proliferation of immature versus mature NK cells.

Transient autoantibodies to danger signals.

The Danger Model predicts that there are some molecules that no immune system can ever be fully tolerant of, namely proteins that are only transiently expressed during times of stress, infection, or injury. Among these are the danger/alarm signals themselves. Accordingly, a fleeting autoantibody response to danger signals is expected during times when they are released. Depending on context, these autoantibodies may serve beneficial "housekeeping" functions by removing surplus danger signals from the circulation or, conversely, create an immunodeficiency. Here, we will focus on the Type 1 Interferons as examples of foreseeable targets for a transient autoantibody response, but the principles outlined should hold for other danger-associated molecules as well.

Autoimmunity: Are we asking the right question?

For decades, the main question immunologists have asked about autoimmunity is "what causes a break in self-tolerance?" We have not found good answers to that question, and I believe we are still so ignorant because it's the wrong question. Rather than a break in self-tolerance, I suggest that many autoimmune diseases might be due to defects in normal tissue physiology.

Neoantigen Presentation and IFNγ Signaling on the Same Tumor-associated Macrophage are Necessary for CD4 T Cell-mediated Antitumor Activity in Mice.

Tumor Associated Macrophages (TAMs) promote tumor survival, angiogenesis and metastases. Although they express MHC Class II molecules, little is known about their ability to present tumor antigens to tumor infiltrating CD4 T cells, nor what are the consequences of such presentation. To answer these questions, we used a C57/BL10 mouse tumor model where we subcutaneously implant a bladder carcinoma cell line naturally expressing the H-Y male antigen into female mice, making the H-Y antigen a de facto neoantigen. We found that TAMs indeed present tumor antigens to effector CD4 T cells and that such presentation is necessary for tumor rejection. As consequence of this interaction TAMs are re-educated to produce lower amounts of tumor promoting proteins and greater amounts of inflammatory proteins. The re-education process of the TAMs is transcriptionally characterized by an IFN-γ signature, including genes of known anti-viral and anti-bacterial functions. CD4 production of IFN-γ, and not TNF-α or CD40L, is required for the re-education process and tumor rejection. Furthermore, IFN-γ signaling on antigen presenting TAMs and not on bystander TAMs, is necessary for the anti-tumor effect. These data identify critical mechanisms of tumor rejection by CD4 T cells and underscores the importance of effector CD4 T cell-tissue macrophage interactions not only at the tumors site but potentially in other tissues.

Are major histocompatibility complex molecules involved in the survival of naive CD4+ T cells?

The exact role of major histocompatibility complex (MHC) molecules in the peripheral survival of naive T cells is controversial, as some studies have suggested that they are critically required whereas others have suggested that they are not. Here we controlled for some of the features that differed among the earlier studies, and analyzed both the survival and expansion of naive CD4+ T cells transferred into MHC syngeneic, allogeneic, or MHC negative environments. We found that naive T cells transferred into MHC negative or allogeneic environments often fail to survive because of rejection and/or competition by natural killer (NK) cells, rather than failure to recognize a particular MHC allele. In the absence of NK cells, naive CD4+ T cells survived equally well regardless of the MHC type of the host. There was, however, an MHC requirement for extensive space-induced "homeostatic" expansion. Although the first few divisions occurred in the absence of MHC molecules, the cells did not continue to divide or transit to a CD44hi phenotype. Surprisingly, this MHC requirement could be satisfied by alleles other than the restricting haplotype. Therefore, space-induced expansion and survival are two different phenomena displaying different MHC requirements. Memory CD4+ T cells, whose survival and expansion showed no requirements for MHC molecules at all, dampened the space-induced expansion of naive cells, showing that the two populations are not independent in their requirements for peripheral niches.

Antigen-specific T-T interactions regulate CD4 T-cell expansion.

The regulation of CD4 T-cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T-cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate major histocompatibility class (MHC)/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way, CD4 T-cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

Strong impact of closing schools, closing bars and wearing masks during the Covid-19 pandemic: results from a simple and revealing analysis.

Many complex mathematical and epidemiological methods have been used to model the Covid-19 pandemic. Among other results from these models has been the view that closing schools had little impact on infection rates in several countries1. We took a different approach. Making one assumption, we simply plotted cases, hospitalizations and deaths, on a log2 Y axis and a linear date-based X axis, and analyzed them using segmented regression, a powerful method that has largely been overlooked during this pandemic. Here we show that the data fit straight lines with correlation coefficients ranging from 92% - 99%, and that these lines broke at interesting intervals, revealing that school closings dropped infection rates in half, lockdowns dropped the rates 3 to 4 fold, and other actions (such as closing bars and mandating masks) brought the rates even further down. Hospitalizations and deaths paralleled cases, with lags of three to ten days. The graphs, which are easy to read, reveal changes in infection rates that are not obvious using other graphing methods, and have several implications for modeling and policy development during this and future pandemics. Overall, other than full lockdowns, three interventions had the most impact: closing schools, closing bars and wearing masks: a message easily understood by the public.

Bone Marrow-Derived Dendritic Cell Cultures from RAG-/- Mice Include IFN-γ-Producing NK Cells.

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as "professional" APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG-/-, but not RAG-/- γc chain-/- mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.

Unexpected regeneration in middle-aged mice.

Complete regeneration of damaged extremities, including both the epithelium and the underlying tissues, is thought to occur mainly in embryos, fetuses, and juvenile mammals, but only very rarely in adult mammals. Surprisingly, we found that common strains of mice are able to regenerate all of the tissues necessary to completely fill experimentally punched ear holes, but only if punched at middle age. Although young postweaning mice regrew the epithelium without typical pre-scar granulation tissue, they showed only minimal regeneration of connective tissues. In contrast, mice punched at 5-11 months of age showed true amphibian-like blastema formation and regrowth of cartilage, fat, and dermis, with blood vessels, sebaceous glands, hair follicles, and, in black mice, melanocytes. These data suggest that at least partial appendage regeneration may be more common in adult mammals than previously thought and call into question the common view that regenerative ability is lost with age. The data suggest that the age at which various inbred mouse strains become capable of epimorphic regeneration may be correlated with adult body weight.

Molecular profiling improves diagnoses of rejection and infection in transplanted organs.

The monitoring of transplanted hearts is currently based on histological evaluation of endomyocardial biopsies, a method that is fairly insensitive and that does not always accurately discriminate between rejection and infection in the heart. Accurate diagnosis of rejection and infection is absolutely crucial, however, as the respective treatments are completely different. Using microarrays, we analyzed gene expression in 76 cardiac biopsies from 40 heart recipients undergoing rejection, no rejection, or Trypanosoma cruzi infection. We found a set of genes whose expression patterns were typical of acute rejection, and another set of genes that discriminated between rejection and T cruzi infection. These sets revealed acute rejection episodes up to 2 weeks earlier, and trypanosome infection up to 2 months earlier than did histological evaluation. When applied to raw data from other institutions, the 2 sets of predictive genes were also able to accurately pinpoint acute rejection of lung and kidney transplants, as well as bacterial infections in kidneys. In addition to their usefulness as diagnostic tools, the data suggest that there are similarities in the biology of the processes involved in rejection of different grafts and also in the tissue responses to pathogens as diverse as bacteria and protozoa.

Mechanisms by which Porphyromonas gingivalis evades innate immunity.

The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines.

Ly6C(hi) Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis.

Antibiotics, though remarkably useful, can also cause certain adverse effects. We detected that treatment of adult mice with antibiotics decreases hippocampal neurogenesis and memory retention. Reconstitution with normal gut flora (SPF) did not completely reverse the deficits in neurogenesis unless the mice also had access to a running wheel or received probiotics. In parallel to an increase in neurogenesis and memory retention, both SPF-reconstituted mice that ran and mice supplemented with probiotics exhibited higher numbers of Ly6C(hi) monocytes in the brain than antibiotic-treated mice. Elimination of Ly6C(hi) monocytes by antibody depletion or the use of knockout mice resulted in decreased neurogenesis, whereas adoptive transfer of Ly6C(hi) monocytes rescued neurogenesis after antibiotic treatment. We propose that the rescue of neurogenesis and behavior deficits in antibiotic-treated mice by exercise and probiotics is partially mediated by Ly6C(hi) monocytes.