Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

ILC3s restrict the dissemination of intestinal bacteria to safeguard liver regeneration after surgery.

It is generally believed that environmental or cutaneous bacteria are the main origin of surgical infections. Therefore, measures to prevent postoperative infections focus on optimizing hygiene and improving asepsis and antisepsis. In a large cohort of patients with infections following major surgery, we identified that the causative bacteria are mainly of intestinal origin. Postoperative infections of intestinal origin were also found in mice undergoing partial hepatectomy. CCR6+ group 3 innate lymphoid cells (ILC3s) limited systemic bacterial spread. Such bulwark function against host invasion required the production of interleukin-22 (IL-22), which controlled the expression of antimicrobial peptides in hepatocytes, thereby limiting bacterial spread. Using genetic loss-of-function experiments and punctual depletion of ILCs, we demonstrate that the failure to restrict intestinal commensals by ILC3s results in impaired liver regeneration. Our data emphasize the importance of endogenous intestinal bacteria as a source for postoperative infection and indicate ILC3s as potential new targets.

The Delicate Skin of Preterm Infants: Barrier Function, Immune-Microbiome Interaction, and Clinical Implications.

The skin of preterm infants is a delicate organ with critical structural and functional differences as compared to term born infants. Unique features contribute to an increased susceptibility to injury, infection, thermal instability, and water loss. During rapid, often accelerated adaption of the physical barrier function of preterm skin, a parallel and mutual development of host skin immunity and skin microbiome seem to be crucial for skin homeostasis. Recent advances in molecular biology have enabled researchers to gain a deeper understanding of the microbial community composition of preterm skin and the important relationship with microbiome composition of other body sites. Nevertheless, several questions remain to be answered, including niche factors and environmental influences on skin maturation. In line with that, evidence-based guidelines on skin care practice in preterm infants are missing. This review articles aims to provide an overview of the current knowledge of preterm infant skin development including immune and barrier function, host-microbial interactions, and potential clinical implications.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Optical imaging of the small intestine immune compartment across scales.

The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography (OPT) enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify the density of villi throughout centimetre-long segments of intestine. Furthermore, we exhibit the age and microbiota dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D imaging with reverse-OPT and high-resolution 2D imaging allows accurate localisation of ROIs and adds value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment.

Lymphatic migration of unconventional T cells promotes site-specific immunity in distinct lymph nodes.

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Intraperitoneal microbial contamination drives post-surgical peritoneal adhesions by mesothelial EGFR-signaling.

Abdominal surgeries are lifesaving procedures but can be complicated by the formation of peritoneal adhesions, intra-abdominal scars that cause intestinal obstruction, pain, infertility, and significant health costs. Despite this burden, the mechanisms underlying adhesion formation remain unclear and no cure exists. Here, we show that contamination of gut microbes increases post-surgical adhesion formation. Using genetic lineage tracing we show that adhesion myofibroblasts arise from the mesothelium. This transformation is driven by epidermal growth factor receptor (EGFR) signaling. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor, are sufficient to induce these changes. Correspondingly, EGFR inhibition leads to a significant reduction of adhesion formation in mice. Adhesions isolated from human patients are enriched in EGFR positive cells of mesothelial origin and human mesothelium shows an increase of mesothelial EGFR expression during bacterial peritonitis. In conclusion, bacterial contamination drives adhesion formation through mesothelial EGFR signaling. This mechanism may represent a therapeutic target for the prevention of adhesions after intra-abdominal surgery.

Microbiota-derived acetate enables the metabolic fitness of the brain innate immune system during health and disease.

As tissue macrophages of the central nervous system (CNS), microglia constitute the pivotal immune cells of this organ. Microglial features are strongly dependent on environmental cues such as commensal microbiota. Gut bacteria are known to continuously modulate microglia maturation and function by the production of short-chain fatty acids (SCFAs). However, the precise mechanism of this crosstalk is unknown. Here we determined that the immature phenotype of microglia from germ-free (GF) mice is epigenetically imprinted by H3K4me3 and H3K9ac on metabolic genes associated with substantial functional alterations including increased mitochondrial mass and specific respiratory chain dysfunctions. We identified acetate as the essential microbiome-derived SCFA driving microglia maturation and regulating the homeostatic metabolic state, and further showed that it is able to modulate microglial phagocytosis and disease progression during neurodegeneration. These findings indicate that acetate is an essential bacteria-derived molecule driving metabolic pathways and functions of microglia during health and perturbation.

The gut microbiota modulates brain network connectivity under physiological conditions and after acute brain ischemia.

The gut microbiome has been implicated as a key regulator of brain function in health and disease. But the impact of gut microbiota on functional brain connectivity is unknown. We used resting-state functional magnetic resonance imaging in germ-free and normally colonized mice under naive conditions and after ischemic stroke. We observed a strong, brain-wide increase of functional connectivity in germ-free animals. Graph theoretical analysis revealed significant higher values in germ-free animals, indicating a stronger and denser global network but with less structural organization. Breakdown of network function after stroke equally affected germ-free and colonized mice. Results from histological analyses showed changes in dendritic spine densities, as well as an immature microglial phenotype, indicating impaired microglia-neuron interaction in germ-free mice as potential cause of this phenomenon. These results demonstrate the substantial impact of bacterial colonization on brain-wide function and extend our so far mainly (sub) cellular understanding of the gut-brain axis.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

Effector differentiation downstream of lineage commitment in ILC1s is driven by Hobit across tissues.

Innate lymphoid cells (ILCs) participate in tissue homeostasis, inflammation, and early immunity against infection. It is unclear how ILCs acquire effector function and whether these mechanisms differ between organs. Through multiplexed single-cell mRNA sequencing, we identified cKit+CD127hiTCF-1hi early differentiation stages of T-bet+ ILC1s. These cells were present across different organs and had the potential to mature toward CD127intTCF-1int and CD127-TCF-1- ILC1s. Paralleling a gradual loss of TCF-1, differentiating ILC1s forfeited their expansion potential while increasing expression of effector molecules, reminiscent of T cell differentiation in secondary lymphoid organs. The transcription factor Hobit was induced in TCF-1hi ILC1s and was required for their effector differentiation. These findings reveal sequential mechanisms of ILC1 lineage commitment and effector differentiation that are conserved across tissues. Our analyses suggest that ILC1s emerge as TCF-1hi cells in the periphery and acquire a spectrum of organ-specific effector phenotypes through a uniform Hobit-dependent differentiation pathway driven by local cues.

Microbiota-derived short chain fatty acids modulate microglia and promote Aß plaque deposition.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aß deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aß plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aß plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aß plaques upon SCFA supplementation, microglia contained less intracellular Aß. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aß deposition likely via modulation of the microglial phenotype.

Long-term evolution and short-term adaptation of microbiota strains and sub-strains in mice.

Isobiotic mice, with an identical stable microbiota composition, potentially allow models of host-microbial mutualism to be studied over time and between different laboratories. To understand microbiota evolution in these models, we carried out a 6-year experiment in mice colonized with 12 representative taxa. Increased non-synonymous to synonymous mutation rates indicate positive selection in multiple taxa, particularly for genes annotated for nutrient acquisition or replication. Microbial sub-strains that evolved within a single taxon can stably coexist, consistent with niche partitioning of ecotypes in the complex intestinal environment. Dietary shifts trigger rapid transcriptional adaptation to macronutrient and micronutrient changes in individual taxa and alterations in taxa biomass. The proportions of different sub-strains are also rapidly altered after dietary shift. This indicates that microbial taxa within a mouse colony adapt to changes in the intestinal environment by long-term genomic positive selection and short-term effects of transcriptional reprogramming and adjustments in sub-strain proportions.

Resident macrophages acquire innate immune memory in staphylococcal skin infection.

Staphylococcus aureus (S. aureus) is a common colonizer of healthy skin and mucous membranes. At the same time, S. aureus is the most frequent cause of skin and soft tissue infections. Dermal macrophages (Mφ) are critical for the coordinated defense against invading S. aureus, yet they have a limited life span with replacement by bone marrow derived monocytes. It is currently poorly understood whether localized S. aureus skin infections persistently alter the resident Mφ subset composition and resistance to a subsequent infection. In a strictly dermal infection model we found that mice, which were previously infected with S. aureus, showed faster monocyte recruitment, increased bacterial killing and improved healing upon a secondary infection. However, skin infection decreased Mφ half-life, thereby limiting the duration of memory. In summary, resident dermal Mφ are programmed locally, independently of bone marrow-derived monocytes during staphylococcal skin infection leading to transiently increased resistance against a second infection.

Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection.

Microbiota-induced tissue signals regulate ILC3-mediated antigen presentation.

Although group 3 innate lymphoid cells (ILC3s) are efficient inducers of T cell responses in the spleen, they fail to induce CD4+ T cell proliferation in the gut. The signals regulating ILC3-T cell responses remain unknown. Here, we show that transcripts associated with MHC II antigen presentation are down-modulated in intestinal natural cytotoxicity receptor (NCR)- ILC3s. Further data implicate microbiota-induced IL-23 as a crucial signal for reversible silencing of MHC II in ILC3s, thereby reducing the capacity of ILC3s to present antigen to T cells in the intestinal mucosa. Moreover, IL-23-mediated MHC II suppression is dependent on mTORC1 and STAT3 phosphorylation in NCR- ILC3s. By contrast, splenic interferon-γ induces MHC II expression and CD4+ T cell stimulation by NCR- ILC3s. Our results thus identify biological circuits for tissue-specific regulation of ILC3-dependent T cell responses. These pathways may have implications for inducing or silencing T cell responses in human diseases.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

NLRP6 Deficiency in CD4 T Cells Decreases T Cell Survival Associated with Increased Cell Death.

The nucleotide-binding oligomerization domain (NOD)-like receptors belong to the family of pattern recognition receptors (PRRs). NOD-like receptors play a role in regulation of innate immune response by recognition of both pathogen-associated molecular patterns that are engulfed during phagocytic process and danger-associated molecular patterns that are mainly byproducts of cell stress mediated response. NOD-like family pyrin domain containing 6 (NLRP6) is one of the 14 pyrin domain-containing receptors. NLRP6 is highly expressed by epithelial and goblet cells to regulate epithelial renewal and mucus production in mice and humans, but its function in T cells is rather unknown. Increased caspase-1 activation and cell death were observed in mouse Nlrp6-deficient T cells following adoptive transfer into Rag2-deficient mice, indicating that Nlrp6 deficiency in CD4+ T cells led to decreased survival.