Lactation-associated macrophages exist in murine mammary tissue and human milk.

Macrophages are involved in immune defense, organogenesis and tissue homeostasis. Macrophages contribute to the different phases of mammary gland remodeling during development, pregnancy and involution postlactation. Less is known about the dynamics of mammary gland macrophages in the lactation stage. Here, we describe a macrophage population present during lactation in mice. By multiparameter flow cytometry and single-cell RNA sequencing, we identified a lactation-induced CD11c+CX3CR1+Dectin-1+ macrophage population (liMac) that was distinct from the two resident F4/80hi and F4/80lo macrophage subsets present pregestationally. LiMacs were predominantly monocyte-derived and expanded by proliferation in situ concomitant with nursing. LiMacs developed independently of IL-34, but required CSF-1 signaling and were partly microbiota-dependent. Locally, they resided adjacent to the basal cells of the alveoli and extravasated into the milk. We found several macrophage subsets in human milk that resembled liMacs. Collectively, these findings reveal the emergence of unique macrophages in the mammary gland and milk during lactation.

A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis.

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response.

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.

Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.

The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.

Microbiota, diet, and the gut-brain axis in multiple sclerosis and stroke.

Intestinal microbiota can influence the phenotype and function of immune cell responses through the dissemination of bacterial antigens or metabolites. Diet is one of the major forces shaping the microbiota composition and metabolism, contributing to host homeostasis and disease susceptibility. Currently, nutrition is a complementary and alternative approach to the management of metabolic and neurological diseases and cancer. However, the knowledge of the exact mechanism of action of diet and microbiota on the gut-brain communication is only developing in recent years. Here, we reviewed the current knowledge on the effect of diet and microbiota on the gut-brain axis in patients with two different central nervous system diseases, multiple sclerosis and stroke. We have also highlighted the open questions in the field that we believe are important to address to gain a deeper understanding of the mechanisms by which diet can directly or indirectly affect the host via the microbiota. We think this will open up new approaches to the treatment, diagnosis, and monitoring of various diseases.

Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

Host-microbiota interactions and adaptive immunity.

All mucosal surfaces are colonized with a vast number of microbes, which are essential for stimulating and regulating the immune system. While intrinsic and innate mechanisms exist to promote a strong barrier between the microbiota and the host to ensure compartmentalization, the microbiota is also able to induce robust adaptive immunity. In this review, we discuss the interplay between the microbiota and the adaptive immune system, with a focus on the induction of mucosal and systemic antibody responses and newly defined roles of maternal antibodies. We also highlight recent studies that aim to decipher microbial antigen-specificity of the T-cell compartment.

Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1ß.

IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However, the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells, we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-γ, but no IL-10, whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6, IL-23 and IL-1ß contributed to TH17 differentiation induced by both pathogens, but IL-1ß was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-γ double-producing cells. In addition, IL-1ß inhibited IL-10 production in differentiating and in memory TH17 cells, whereas blockade of IL-1ß in vivo led to increased IL-10 production by memory TH17 cells. We also show that, after restimulation, TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-γt. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-γ or IL-10, and identify IL-1ß and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase.

Detrimental effect of systemic antimicrobial CD4+ T-cell reactivity on gut epithelial integrity.

Healthy host-microbe mutualism relies on compartmentalization and proper regulation of systemic and mucosal immune responses. Nevertheless, the systemic immune system is frequently exposed to bouts of bacteraemia, which can trigger systemic antimicrobial immune reactivity including CD4+ T cells. Low-level bacteraemia can occur when immune compartmentalization is compromised, for example in the presence of innate immune deficiency or following use of non-steroidal anti-inflammatory drugs. We generated an Escherichia coli strain expressing a defined T helper neo-epitope to study systemic antigen-specific antimicrobial CD4+ T cells and their potential involvement in the pathogenisis of inflammatory bowel diseases. We found that the dose of bacteria required for the induction of systemic antimicrobial CD4+ T-cell proliferation was high and not easily reached under physiological conditions. Importantly, however, when intestinal barrier function was compromised by induced damage to the intestinal epithelium, the presence of systemic antimicrobial CD4+ T cells specific for a single neo-antigen resulted in dramatically increased levels of bacterial translocation. This study therefore demonstrates that systemic antimicrobial CD4+ T-cell reactivity might impact adversely on the mucosa under conditions of reduced barrier function and that despite strong mucosal immune regulation, antigen-specific recognition is still sensitive.

Reproductive allocation in pulsed-resource environments: a comparative study in two populations of wild boar.

Pulsed resources influence the demography and evolution of consumer populations and, by cascading effect, the dynamics of the entire community. Mast seeding provides a case study for exploring the evolution of life history traits of consumers in fluctuating environments. Wild boar (Sus scrofa) population dynamics is related to seed availability (acorns/beechnuts). From a long-term monitoring of two populations subjected to markedly different environmental contexts (i.e., both low vs. high frequency of pulsed resources and low vs. high hunting pressure in Italy and in France, respectively), we assessed how pulsed resources shape the reproductive output of females. Using path analyses, we showed that in both populations, abundant seed availability increases body mass and both the absolute and the relative (to body mass) allocation to reproduction through higher fertility. In the Italian population, females equally relied on past and current resources for reproduction and ranked at an intermediate position along the capital-income continuum of breeding tactics. In contrast, in the French population, females relied on current more than past resources and ranked closer to the income end of the continuum. In the French population, one-year old females born in acorn-mast years were heavier and had larger litter size than females born in beechnut-mast years. In addition to the quantity, the type of resources (acorns/beechnuts) has to be accounted for to assess reliably how females allocate resources to reproduction. Our findings highlight a high plasticity in breeding tactics in wild boar females and provide new insight on allocation strategies in fluctuating environments.

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response.

IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naive CD4+ T cells.

The interleukin-1 (IL-1) superfamily of cytokines comprises a set of pivotal mediators of inflammation. Among them, the action of IL-36 cytokines in immune responses has remained elusive. In a recent study, we demonstrated a direct effect of IL-36 on immune cells. Here we show that, among T cells, the IL-36 receptor is predominantly expressed on naive CD4(+) T cells and that IL-36 cytokines act directly on naive T cells by enhancing both cell proliferation and IL-2 secretion. IL-36ß acts in synergy with IL-12 to promote Th1 polarization and IL-36 signaling is also involved in mediating Th1 immune responses to Bacillus Calmette-Guerin infection in vivo. Our findings point toward a critical function of IL-36 in the priming of Th1 cell responses in vitro, and in adaptive immunity in a model of mycobacterial infection in vivo.

Nutrition during pregnancy: Influence on the gut microbiome and fetal development.

Pregnancy is a finely tuned process, with the health and well-being of the developing fetus determined by the metabolic status and dietary intake of the mother. The maternal gut microbiome is remodeled during pregnancy, and this, coupled with the maternal nutrient intake during gestation shapes the production of metabolites that can cross the placenta and affect fetal development. As posited by the Developmental Origins of Health and Disease Hypothesis, such environmental influences can have major effects on the developing organ systems. When occurring at particularly sensitive gestational time points, these developmental programming events can have long lasting effects on offspring adaptation to the postnatal environment, and major health implications later in life. This review will summarize current knowledge on how pregnancy and maternal dietary intake intrinsically and extrinsically modify maternal gut microbiota composition and metabolite production. Further, we will assess how these factors shape the fetal landscape and ultimately contribute to offspring health. DOHaD, fetal development, metabolites, microbiome, nutrition, pregnancy, short-chain fatty acids.

The impact of microbiota and ketogenic diet interventions in the management of drug-resistant epilepsy.

AIM: Drug-resistant epilepsy (DRE) is a neurological disorder characterized by uncontrolled seizures. It affects between 10%-40% of the patients with epilepsy worldwide. Drug-resistant patients have been reported to have a different microbiota composition compared to drug-sensitive patients and healthy controls. Importantly, fecal microbiota transplantations (FMTs), probiotic and dietary interventions have been shown to be able to reduce seizure frequency and improve the quality of life in drug-resistant patients. The classic ketogenic diet (KD) and its modifications may reduce seizures in DRE in some patients, whereas in others they do not. The mechanisms mediating the dietary effects remain elusive, although it is known that gut microbes play an important role in transmitting dietary effects to the host. Indeed, specific commensal microbes differ even between responders and non-responders to KD treatment. METHODS: In this narrative mini-review, we summarize what is known about the gut microbiota changes and ketogenic diets with special focus on patients with DRE. RESULTS AND CONCLUSIONS: By highlighting unanswered questions and by suggesting future research directions, we map the route towards future improvement of successful DRE therapy.

IL-36R ligands are potent regulators of dendritic and T cells.

IL-36α (IL-1F6), IL-36ß (IL-1F8), and IL-36γ (IL-1F9) are members of the IL-1 family of cytokines. These cytokines bind to IL-36R (IL-1Rrp2) and IL-1RAcP, activating similar intracellular signals as IL-1, whereas IL-36Ra (IL-1F5) acts as an IL-36R antagonist (IL-36Ra). In this study, we show that both murine bone marrow-derived dendritic cells (BMDCs) and CD4(+) T lymphocytes constitutively express IL-36R and respond to IL-36α, IL-36ß, and IL-36γ. IL-36 induced the production of proinflammatory cytokines, including IL-12, IL-1ß, IL-6, TNF-α, and IL-23 by BMDCs with a more potent stimulatory effect than that of other IL-1 cytokines. In addition, IL-36ß enhanced the expression of CD80, CD86, and MHC class II by BMDCs. IL-36 also induced the production of IFN-γ, IL-4, and IL-17 by CD4(+) T cells and cultured splenocytes. These stimulatory effects were antagonized by IL-36Ra when used in 100- to 1000-fold molar excess. The immunization of mice with IL-36ß significantly and specifically promoted Th1 responses. Our data thus indicate a critical role of IL-36R ligands in the interface between innate and adaptive immunity, leading to the stimulation of T helper responses.

Plastics in our ocean as transdisciplinary challenge.

This conference report summarizes the current challenges of researching microplastics pollution in the ocean as debated by international experts and stakeholders at a workshop held in San Sebastián, Spain, 1-2 October 2019. The transdisciplinary, co-learning approach of this report stressed the need to incorporate multiple perspective in solving the problem of microplastics and resulted in three proposed actions: (i) filtering microplastics from waste waters; (ii) mandatory ecolabels on plastic products packages; and (iii) circular economy of packaging plastics.

Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice.

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.

The Gut-Brain Axis: How Microbiota and Host Inflammasome Influence Brain Physiology and Pathology.

The human microbiota has a fundamental role in host physiology and pathology. Gut microbial alteration, also known as dysbiosis, is a condition associated not only with gastrointestinal disorders but also with diseases affecting other distal organs. Recently it became evident that the intestinal bacteria can affect the central nervous system (CNS) physiology and inflammation. The nervous system and the gastrointestinal tract are communicating through a bidirectional network of signaling pathways called the gut-brain axis, which consists of multiple connections, including the vagus nerve, the immune system, and bacterial metabolites and products. During dysbiosis, these pathways are dysregulated and associated with altered permeability of the blood-brain barrier (BBB) and neuroinflammation. However, numerous mechanisms behind the impact of the gut microbiota in neuro-development and -pathogenesis remain poorly understood. There are several immune pathways involved in CNS homeostasis and inflammation. Among those, the inflammasome pathway has been linked to neuroinflammatory conditions such as multiple sclerosis, Alzheimer's and Parkinson's diseases, but also anxiety and depressive-like disorders. The inflammasome complex assembles upon cell activation due to exposure to microbes, danger signals, or stress and lead to the production of pro-inflammatory cytokines (interleukin-1ß and interleukin-18) and to pyroptosis. Evidences suggest that there is a reciprocal influence of microbiota and inflammasome activation in the brain. However, how this influence is precisely working is yet to be discovered. Herein, we discuss the status of the knowledge and the open questions in the field focusing on the function of intestinal microbial metabolites or products on CNS cells during healthy and inflammatory conditions, such as multiple sclerosis, Alzheimer's and Parkinson's diseases, and also neuropsychiatric disorders. In particular, we focus on the innate inflammasome pathway as immune mechanism that can be involved in several of these conditions, upon exposure to certain microbes.

Female mating tactics in lekking fallow deer (Dama dama): experience explains inter-individual variability more than costs.

Most studies on ungulate reproduction have focused on the covariates of male reproductive success, while there is much less information on female tactics of mate choice. The aim of this work is to fill this gap and to assess condition-dependent variations in female tactics in a lekking fallow deer (Dama dama) population. In particular, we investigated three indirect selection mechanisms: i) aggregation: when females join an already formed female group; ii) copying: when females copy the mate choice of other females and iii) territory choice: when females select a territory where many copulations had previously occurred. Our results show that female fallow deer, which are less experienced (younger) and/or incur higher travel costs (home range far from the lek), adopt indirect forms of mate selection more often than older females or females residing near the lek, respectively. Compared to adults, younger females remained longer in the lek (almost three times) and in male territories, returning to the lek after copulation. However, despite the time spent at the lek, younger females were not able to select the highest-rank males, and relied on territory choice more often than older females. Farther does visited the lek less frequently (farthest females only once) and arrived on average 5 days later than closer females (which performed up to 7 visits), but they were seen more often within female groups (aggregation). We did not find a different amount of copying in younger or in farther females. Our results contribute to advance our understanding of female behaviours in ungulate leks.

Evaluation of primer pairs for microbiome profiling from soils to humans within the One Health framework.

The 'One Health' framework emphasizes the ecological relationships between soil, plant, animal and human health. Microbiomes play important roles in these relationships, as they modify the health and performance of the different compartments and influence the transfer of energy, matter and chemicals between them. Standardized methods to characterize microbiomes along food chains are, however, currently lacking. To address this methodological gap, we evaluated the performance of DNA extraction kits and commonly recommended primer pairs targeting different hypervariable regions (V3-V4, V4, V5-V6, V5-V6-V7) of the 16S rRNA gene, on microbiome samples along a model food chain, including soils, maize roots, cattle rumen, and cattle and human faeces. We also included faeces from gnotobiotic mice colonized with defined bacterial taxa and mock communities to confirm the robustness of our molecular and bioinformatic approaches on these defined low microbial diversity samples. Based on Amplicon Sequence Variants, the primer pair 515F-806R led to the highest estimates of species richness and diversity in all sample types and offered maximum diversity coverage of reference databases in in silico primer analysis. The influence of the DNA extraction kits was negligible compared to the influence of the choice of primer pairs. Comparing microbiomes using 515F-806R revealed that soil and root samples have the highest estimates of species richness, while lowest richness was observed in human faeces. Primer pair choice directly influenced the estimation of community changes within and across compartments and may give rise to preferential detection of specific taxa. This work demonstrates why a standardized approach is necessary to analyse microbiomes within and between source compartments along food chains in the context of the One Health framework.