Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Integrating single-cell multi-omics and prior biological knowledge for a functional characterization of the immune system.

The immune system comprises diverse specialized cell types that cooperate to defend the host against a wide range of pathogenic threats. Recent advancements in single-cell and spatial multi-omics technologies provide rich information about the molecular state of immune cells. Here, we review how the integration of single-cell and spatial multi-omics data with prior knowledge-gathered from decades of detailed biochemical studies-allows us to obtain functional insights, focusing on gene regulatory processes and cell-cell interactions. We present diverse applications in immunology and critically assess underlying assumptions and limitations. Finally, we offer a perspective on the ongoing technological and algorithmic developments that promise to get us closer to a systemic mechanistic understanding of the immune system.

Intestinal damage is required for the pro-inflammatory differentiation of commensal CBir1-specific T cells.

Commensal-specific clusters of differentiation (CD)4+ T cells are expanded in patients with inflammatory bowel disease (IBD) compared to healthy individuals. How and where commensal-specific CD4+ T cells get activated is yet to be fully understood. We used CBir1 TCR-transgenic CD4+ T cells, specific to a commensal bacterial antigen, and different mouse models of IBD to characterize the dynamics of commensal-specific CD4+ T-cells activation. We found that CBir1 T cells proliferate following intestinal damage and cognate antigen presentation is mediated by CD11c+ cells in the colon-draining mesenteric lymph nodes. Using assay for transposase-accessible chromatin sequencing and flow cytometry, we showed that activated CBir1 T cells preferentially acquire an effector rather than regulatory phenotype, which is plastic over time. Moreover, CBir1 T cells, while insufficient to initiate intestinal inflammation, contributed to worse disease outcomes in the presence of other CD4+ T cells. Our results suggest that the commensal-specific T-cell responses observed in IBD exacerbate rather than initiate disease.

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms.

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400-650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

Amerindian ancestry proportion as a risk factor for inflammatory bowel diseases: results from a Latin American Andean cohort.

Background and aims: Latin American populations remain underrepresented in genetic studies of inflammatory bowel diseases (IBDs). Most genetic association studies of IBD rely on Caucasian, African, and Asian individuals. These associations have yet to be evaluated in detail in the Andean region of South America. We explored the contribution of IBD-reported genetic risk variants to a Chilean cohort and the ancestry contribution to IBD in this cohort. Methods: A total of 192 Chilean IBD patients were genotyped using Illumina's Global Screening Array. Genotype data were combined with similar information from 3,147 Chilean controls. The proportions of Aymara, African, European, and Mapuche ancestries were estimated using the software ADMIXTURE. We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) for gender, age, and ancestry proportions. We also explored associations with previously reported IBD-risk variants independently and in conjunction with genetic ancestry. Results: The first and third quartiles of the proportion of Mapuche ancestry in IBD patients were 24.7 and 34.2%, respectively, and the corresponding OR was 2.30 (95%CI 1.52-3.48) for the lowest vs. the highest group. Only one variant (rs7210086) of the 180 reported IBD-risk SNPs was associated with IBD risk in the Chilean cohort (adjusted P = 0.01). This variant is related to myeloid cells. Conclusion: The type and proportion of Native American ancestry in Chileans seem to be associated with IBD risk. Variants associated with IBD risk in this Andean region were related to myeloid cells and the innate immune response.

Short-term dietary changes can result in mucosal and systemic immune depression.

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.

Low-grade intestinal inflammation two decades after pelvic radiotherapy.

BACKGROUND: Radiotherapy is effective in the treatment of cancer but also causes damage to non-cancerous tissue. Pelvic radiotherapy may produce chronic and debilitating bowel symptoms, yet the underlying pathophysiology is still undefined. Most notably, although pelvic radiotherapy causes an acute intestinal inflammation there is no consensus on whether the late-phase pathophysiology contains an inflammatory component or not. To address this knowledge gap, we examined the potential presence of a chronic inflammation in mucosal biopsies from irradiated pelvic cancer survivors. METHODS: We biopsied 24 cancer survivors two to 20 years after pelvic radiotherapy, and four non-irradiated controls. Using tandem mass tag (TMT) mass spectrometry and mRNA sequencing (mRNA-seq), we charted proteomic and transcriptomic profiles of the mucosal tissue previously exposed to a high or a low/no dose of radiation. Changes in the immune cell populations were determined with flow cytometry. The integrity of the protective mucus layers were determined by permeability analysis and 16S rRNA bacterial detection. FINDINGS: 942 proteins were differentially expressed in mucosa previously exposed to a high radiation dose compared to a low radiation dose. The data suggested a chronic low-grade inflammation with neutrophil activity, which was confirmed by mRNA-seq and flow cytometry and further supported by findings of a weakened mucus barrier with bacterial infiltration. INTERPRETATION: Our results challenge the idea that pelvic radiotherapy causes an acute intestinal inflammation that either heals or turns fibrotic without progression to chronic inflammation. This provides a rationale for exploring novel strategies to mitigate chronic bowel symptoms in pelvic cancer survivors. FUNDING: This study was supported by the King Gustav V Jubilee Clinic Cancer Foundation (CB), The Adlerbertska Research Foundation (CB), The Swedish Cancer Society (GS), The Swedish State under the ALF agreement (GS and CB), Mary von Sydow's foundation (MA and VP).

The single-cell transcriptional landscape of innate and adaptive lymphocytes in pediatric-onset colitis.

Innate lymphoid cells (ILCs) are considered innate counterparts of adaptive T cells; however, their common and unique transcriptional signatures in pediatric inflammatory bowel disease (pIBD) are largely unknown. Here, we report a dysregulated colonic ILC composition in pIBD colitis that correlates with inflammatory activity, including accumulation of naive-like CD45RA+CD62L- ILCs. Weighted gene co-expression network analysis (WGCNA) reveals modules of genes that are shared or unique across innate and adaptive lymphocytes. Shared modules include genes associated with activation/tissue residency, naivety/quiescence, and antigen presentation. Lastly, nearest-neighbor-based analysis facilitates the identification of "most inflamed" and "least inflamed" lymphocytes in pIBD colon with unique transcriptional signatures. Our study reveals shared and unique transcriptional signatures of colonic ILCs and T cells in pIBD. We also provide insight into the transcriptional regulation of colonic inflammation, deepening our understanding of the potential mechanisms involved in pIBD.

Golgi organization is a determinant of stem cell function in the small intestine.

Cell-to-cell signalling between niche and stem cells regulates tissue regeneration. While the identity of many mediating factors is known, it is largely unknown whether stem cells optimize their receptiveness to niche signals according to the niche organization. Here, we show that Lgr5+ small intestinal stem cells (ISCs) regulate the morphology and orientation of their secretory apparatus to match the niche architecture, and to increase transport efficiency of niche signal receptors. Unlike the progenitor cells lacking lateral niche contacts, ISCs orient Golgi apparatus laterally towards Paneth cells of the epithelial niche, and divide Golgi into multiple stacks reflecting the number of Paneth cell contacts. Stem cells with a higher number of lateral Golgi transported Epidermal growth factor receptor (Egfr) with a higher efficiency than cells with one Golgi. The lateral Golgi orientation and enhanced Egfr transport required A-kinase anchor protein 9 (Akap9), and was necessary for normal regenerative capacity in vitro . Moreover, reduced Akap9 in aged ISCs renders ISCs insensitive to niche-dependent modulation of Golgi stack number and transport efficiency. Our results reveal stem cell-specific Golgi complex configuration that facilitates efficient niche signal reception and tissue regeneration, which is compromised in the aged epithelium.

Metagenomic and single-cell RNA-Seq survey of the Helicobacter pylori-infected stomach in asymptomatic individuals.

Helicobacter pylori colonization of the gastric niche can persist for years in asymptomatic individuals. To deeply characterize the host-microbiota environment in H. pylori-infected (HPI) stomachs, we collected human gastric tissues and performed metagenomic sequencing, single-cell RNA-Seq (scRNA-Seq), flow cytometry, and fluorescent microscopy. HPI asymptomatic individuals had dramatic changes in the composition of gastric microbiome and immune cells compared with noninfected individuals. Metagenomic analysis uncovered pathway alterations related to metabolism and immune response. scRNA-Seq and flow cytometry data revealed that, in contrast to murine stomachs, ILC2s are virtually absent in the human gastric mucosa, whereas ILC3s are the dominant population. Specifically, proportion of NKp44+ ILC3s out of total ILCs were highly increased in the gastric mucosa of asymptomatic HPI individuals, and correlated with the abundance of selected microbial taxa. In addition, CD11c+ myeloid cells and activated CD4+ T cells and B cells were expanded in HPI individuals. B cells of HPI individuals acquired an activated phenotype and progressed into a highly proliferating germinal-center stage and plasmablast maturation, which correlated with the presence of tertiary lymphoid structures within the gastric lamina propria. Our study provides a comprehensive atlas of the gastric mucosa-associated microbiome and immune cell landscape when comparing asymptomatic HPI and uninfected individuals.

Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH.

Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here we develop a method named Enhanced ELectric Fluorescence in situ Hybridization (EEL FISH) that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down toward the surface, preserving single-cell resolution. We apply EEL on eight entire sagittal sections of the mouse brain and measure the expression patterns of up to 440 genes to reveal complex tissue organization. Moreover, EEL can be used to study challenging human samples by removing autofluorescent lipofuscin, enabling the spatial transcriptome of the human visual cortex to be visualized. We provide full hardware specifications, all protocols and complete software for instrument control, image processing, data analysis and visualization.

B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing.

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.

Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine.

Cytokines are immunomodulatory proteins that orchestrate cellular networks in health and disease. Among these, interleukin (IL)-10 is critical for the establishment of intestinal homeostasis, as mutations in components of the IL-10 signaling pathway result in spontaneous colitis. Whether IL-10 plays other than immunomodulatory roles in the intestines is poorly understood. Here, we report that il10, il10ra, and il10rb are expressed in the zebrafish developing intestine as early as 3 days post fertilization. CRISPR/Cas9-generated il10-deficient zebrafish larvae showed an increased expression of pro-inflammatory genes and an increased number of intestinal goblet cells compared to WT larvae. Mechanistically, Il10 promotes Notch signaling in zebrafish intestinal epithelial cells, which in turn restricts goblet cell expansion. Using murine organoids, we showed that IL-10 modulates goblet cell frequencies in mammals, suggesting conservation across species. This study demonstrates a previously unappreciated IL-10-Notch axis regulating goblet cell homeostasis in the developing zebrafish intestine and may help explain the disease severity of IL-10 deficiency in the intestines of mammals.

Mechanisms of mucosal healing: treating inflammatory bowel disease without immunosuppression?

Almost all currently available treatments for inflammatory bowel disease (IBD) act by inhibiting inflammation, often blocking specific inflammatory molecules. However, given the infectious and neoplastic disease burden associated with chronic immunosuppressive therapy, the goal of attaining mucosal healing without immunosuppression is attractive. The absence of treatments that directly promote mucosal healing and regeneration in IBD could be linked to the lack of understanding of the underlying pathways. The range of potential strategies to achieve mucosal healing is diverse. However, the targeting of regenerative mechanisms has not yet been achieved for IBD. Stem cells provide hope as a regenerative treatment and are used in limited clinical situations. Growth factors are available for the treatment of short bowel syndrome but have not yet been applied in IBD. The therapeutic application of organoid culture and stem cell therapy to generate new intestinal tissue could provide a novel mechanism to restore barrier function in IBD. Furthermore, blocking key effectors of barrier dysfunction (such as MLCK or damage-associated molecular pattern molecules) has shown promise in experimental IBD. Here, we review the diversity of molecular targets available to directly promote mucosal healing, experimental models to identify new potential pathways and some of the anticipated potential therapies for IBD.

Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity.

Goblet cells secrete mucin to create a protective mucus layer against invasive bacterial infection and are therefore essential for maintaining intestinal health. However, the molecular pathways that regulate goblet cell function remain largely unknown. Although GPR35 is highly expressed in colonic epithelial cells, its importance in promoting the epithelial barrier is unclear. In this study, we show that epithelial Gpr35 plays a critical role in goblet cell function. In mice, cell-type-specific deletion of Gpr35 in epithelial cells but not in macrophages results in goblet cell depletion and dysbiosis, rendering these animals more susceptible to Citrobacter rodentium infection. Mechanistically, scRNA-seq analysis indicates that signaling of epithelial Gpr35 is essential to maintain normal pyroptosis levels in goblet cells. Our work shows that the epithelial presence of Gpr35 is a critical element for the function of goblet cell-mediated symbiosis between host and microbiota.

The spatial transcriptomic landscape of the healing mouse intestine following damage.

The intestinal barrier is composed of a complex cell network defining highly compartmentalized and specialized structures. Here, we use spatial transcriptomics to define how the transcriptomic landscape is spatially organized in the steady state and healing murine colon. At steady state conditions, we demonstrate a previously unappreciated molecular regionalization of the colon, which dramatically changes during mucosal healing. Here, we identified spatially-organized transcriptional programs defining compartmentalized mucosal healing, and regions with dominant wired pathways. Furthermore, we showed that decreased p53 activation defined areas with increased presence of proliferating epithelial stem cells. Finally, we mapped transcriptomics modules associated with human diseases demonstrating the translational potential of our dataset. Overall, we provide a publicly available resource defining principles of transcriptomic regionalization of the colon during mucosal healing and a framework to develop and progress further hypotheses.

The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy.

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry-based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo. Significance: The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic.

Intestinal helminth infection transforms the CD4+ T cell composition of the skin.

Intestinal helminth parasites can alter immune responses to vaccines, other infections, allergens and autoantigens, implying effects on host immune responses in distal barrier tissues. We herein show that the skin of C57BL/6 mice infected with the strictly intestinal nematode Heligmosomoides polygyrus contain higher numbers of CD4+ T cells compared to the skin of uninfected controls. Accumulated CD4+ T cells were H. polygyrus-specific TH2 cells that skewed the skin CD4+ T cell composition towards a higher TH2/TH1 ratio which persisted after worm expulsion. Accumulation of TH2 cells in the skin was associated with increased expression of the skin-homing chemokine receptors CCR4 and CCR10 on CD4+ T cells in the blood and mesenteric lymph nodes draining the infected intestine and was abolished by FTY720 treatment during infection, indicating gut-to-skin trafficking of cells. Remarkably, skin TH2 accumulation was associated with impaired capacity to initiate IFN-γ recall responses and develop skin-resident memory cells to mycobacterial antigens, both during infection and months after deworming therapy. In conclusion, we show that infection by a strictly intestinal helminth has long-term effects on immune cell composition and local immune responses to unrelated antigens in the skin, revealing a novel process for T cell colonisation and worm-mediated immunosuppression in this organ.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

Perfluorooctanesulfonic acid modulates barrier function and systemic T-cell homeostasis during intestinal inflammation.

The intestinal epithelium is continuously exposed to deleterious environmental factors that might cause aberrant immune responses leading to inflammatory disorders. However, what environmental factors might contribute to disease are poorly understood. Here, to overcome the lack of in vivo models suitable for screening of environmental factors, we used zebrafish reporters of intestinal inflammation. Using zebrafish, we interrogated the immunomodulatory effects of polyfluoroalkyl substances, which have been positively associated with ulcerative colitis incidence. Exposure to perfluorooctanesulfonic acid (PFOS) during 2,4,6-trinitro-benzene sulfonic acid (TNBS)-induced inflammation enhanced the expression of proinflammatory cytokines as well as neutrophil recruitment to the intestine of zebrafish larvae, which was validated in the TNBS-induced colitis mouse model. Moreover, PFOS exposure in mice undergoing colitis resulted in neutrophil-dependent increased intestinal permeability and enhanced PFOS translocation into the circulation. This was associated with a neutrophil-dependent expansion of systemic CD4+ T cells. Thus, our results indicate that PFOS worsens inflammation-induced intestinal damage with disruption of T-cell homeostasis beyond the gut and provides a novel in vivo toolbox to screen for pollutants affecting intestinal homeostasis.