Glioblastoma (GBM) is the most common and malignant brain tumor in adult humans. Recent studies have demonstrated a link between the composition of the gut microbiota and glioma progression. Here, we describe that the growth of glioma in mice is inversely correlated with the relative abundance of the anaerobic bacterium Muribaculum intestinale in the feces. We found that M. intestinale administration: 1) induced an inflammatory environment in the gut; 2) reduced glioma growth; 3) increased the pro-inflammatory profile of tumor-associated microglial cells and the frequency of CD8+ T cells; and 4) increased the peripheral TNF-α levels. The effects induced by M. intestinale administration were significantly attenuated upon toll-like receptor 2 (TLR2) silencing using TLR2-targeting siRNA. As a pattern-recognition receptor, TLR2 detects microbial-associated molecular patterns and orchestrates host immune responses to infection. Collectively, these data demonstrate that M. intestinale induces a pro-inflammatory response in glioma bearing mice, inhibiting tumor growth via TLR2-dependent signaling.

Figure S3. Neutrophils drive the expression of IL-23 by BMDMs. Related to Figure 3. A) Total number of ulcers in the colon of DSS-treated Csfr3+/+ (n=6) and Csf3r-/- (n=7) mice. B) IL-23 levels detected by ELISA in supernatants of BMDMs, neutrophils (NΦ) and BMDM-NΦ co-cultures after stimulation with GM-CSF+CpG. In indicated conditions neutrophils were pre-treated with diphenyleneiodonium (DPI) (10µM) or BMDM-NΦ were cultured in transwells (for all conditions n=6). C) Percentage of viable neutrophils (AnnexinV-/PI-) cultured alone or after co-culture with BMDMs. D) Schematic representation of the treatment schedule for anti-IL-22 treatment and neutrophil adoptive cell transfer during acute colitis. Days of treatment are indicated by red arrows. E) Percentage of body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/-(n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils treated with anti-IL-22 or isotype control (50µg/mouse) via i.p. injection (Csf3r-/- mice+Isotype n=3; Csf3r-/-+anti-IL-22 n=3). A) Representative data of five independent experiments. B) Representative data of three independent experiments. C) Representative of two independent experiments. E) One experiment. A) Unpaired Student’s t-Test. B-C) Multiple Student’s t-Test. E) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Figure S7. A) Frequency of CD4+ Foxp3+ regulatory T cells in colon LP of DSS-treated Csf3r+/+ and Csf3r-/-. Unpaired Student’s t-Test. Data are mean ± SEM. ns: not significant.

Supplementary Table 1. Colitis grade score.

CRISPR-mediated gene editing enables efficient genetic manipulation of ILC2s through ex vivo or in vivo activation and Cas9 RNP delivery. This platform provides a robust approach to dissect gene function in ILC2s, with minimal manipulation.

Figure S5. γδ T cell subsets activation in DSS-treated mice. Related to Figure 4-5 A) Representative gating strategy for γδ T cell subsets in colon LP. B) Frequency of γδ T cell subsets from colon tissue of DSS-treated mice in colon LP. C) AhR expression in γδ T cells subset from colon LP of DSS-treated mice. D-E) Expression of IL-22 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (D) and PMA plus ionomycin (E) analyzed by flow cytometry. F-G) Expression of IL-17 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (F) and PMA plus ionomycin (G) analyzed by flow cytometry. H) RorγT expression in γδ T cell subsets from colon LP of DSS-treated mice. I) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/- (n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils (NΦ) (n=4) with and without antibiotic (ABX) oral treatment (Csf3r+/++ABX n=4 and Csf3r-/-+ABX n=4, Csf3r-/-+NΦ+ABX n=4). Red arrows indicated days of neutrophils transfer. A-H) Representative data of two independent experiments, Csf3r+/+(n=4), Csf3r-/- (n=4). I) One experiments. B-H) Multiple t-test. I) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Sleep pressure reflects the brain's homeostatic need to sleep, but the mechanisms underlying its regulation remain poorly understood. In mice, a subset of cortical inhibitory neuronal nitric oxide synthase (nNOS) positive interneurons tunes the electroencephalographic slow wave activity in the delta band (<4 Hz), marker of sleep pressure. Here, we demonstrate that in mice the natural killer (NK) cells and innate lymphoid cells (ILC)1 depletion inhibits nNOS+ interneurons and EEG delta activity reducing the time spent in the non-rapid eye movement (NREM) sleep. The optogenetic re-activation of nNOS+ interneurons in the cingulate cortex of NK cell/ILC1-depleted mice rescues the EEG delta activity, confirming the link between innate immune cells-nNOS+ interneurons-sleep pressure. Finally, we demonstrated that meningeal NK/ILC1 cells produce IFN-γ in a circadian independent manner and that IFN-γ blockade in vivo mimics the effect of NK cell depletion in mice. These findings provide insights into the complex network involved in sleep regulation and further support the contribution of the innate immune system on sleep pressure.

Figure S2. Neutrophil deficiency is associated with intestinal dysbiosis. Related to Figure 2 A) Representative histological images of H&E-stained colon section from Csf3r+/+(left) and Csf3r-/- (right) mice, after one cycle of DSS, showing the increased inflammatory infiltrate and the presence of bacterial accretions (indicated by black arrow) in Csf3r-/-. B-C) Shannon Index representing the evenness in the overall number of bacterial species between wild-type and neutrophil deficient mice untreated (B) and DSS-treated mice (C). D-E) Taxonomic analysis of the phyla that compose the microbiota of feces of untreated (D) and DSS-treated (E) Csf3r+/+ (untreated n=4, DSS-treated n=3) and Csf3r-/- (untreated n=4, DSS-treated n=4) mice. F) Microbiota depletion efficiency measured by qPCR for 16S gene: complete depletion is achieved after 20 days of antibiotic treatment. G) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=11) and Csf3r-/- (n=9) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5). H) Body weight loss of AOM/DSS treated Csf3r+/+(n=7) and Csf3r-/- (n=5) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5); I) Macroscopic polyp count at the experimental endpoint. A-I) One experiment. B-C) Unpaired Student’s t-Test. F, I) Multiple Student’s t-Test. G-H) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Innate lymphoid cells (ILCs) are key regulators of early immune responses and play a central role in mucosal immunity, where they contribute to host defense and tissue homoeostasis. This review synthesizes evidence that ILCs, including natural killer cells (NKs), ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer (LTi) cells, directly sense pathogens via pattern recognition receptors (PRRs). Beyond their established role as cytokine responders, emerging data reveal that ILCs engage PRRs to initiate complementary, context-dependent signaling pathways. This direct recognition mechanism redefines the functional landscape of ILCs in early immune surveillance, moving beyond reliance on indirect stromal signals. Collectively, these insights reposition ILCs as active sentinels in host defense and highlight the ILC-PRR axis as a novel therapeutic avenue for modulating immune responses in infectious, inflammatory, and cancer-related diseases.

Mast cells (MCs) are multifunctional immune cells with context-dependent functions in cancer. In colorectal cancer (CRC), their contribution remains debated, suggesting that distinct MC subsets may either support tumor progression or promote anti-tumor immunity. Using single-cell RNA sequencing in a mouse model of inflammation-driven CRC, we uncovered extensive MC plasticity during tumor progression. Transcriptomic profiling and pseudotime trajectory analysis revealed a transition from precursor-like MCs exclusively present in adjacent tissue to differentiated tumor-associated MC (TAMC) subsets. TAMCs displayed a distinct repertoire of proteases and pro-inflammatory cytokines, contributing to increased vascular permeability and recruitment of additional immune cells. Moreover, TAMCs exhibited upregulation of molecules with immunosuppressive potential and underwent a tumor microenvironment (TME)-driven metabolic reprogramming. Accordingly, antibody-mediated MC depletion reduced tumor burden. Notably, most of the transcriptional features identified in a murine CRC model were recapitulated in human CRC, supporting the translational relevance of this MC program. Our findings identify a tumor-adapted MC state that orchestrates immune evasion and tissue remodeling during CRC progression, supporting the notion that MC are reprogrammed toward an immune-suppressive and pro-tumorigenic phenotype.

Innate immune cells respond rapidly to environmental cues through signal-regulated transcription factors (SRTFs) that sense changes in the tissue microenvironment. Signal transducer and activator of transcription (STAT) proteins are critical regulators of cytokine signaling and determine polarized immune responses. Herein, we reveal that activated type 2 innate lymphocytes (ILC2s) express STAT4, a SRTF canonically linked to type 1 immunity. STAT4 expression is induced in ILC2s upon activation by the alarmin IL-25 and linked with accumulation of lung inflammatory ILC2s (iILC2s). Despite elevated STAT4 expression, iILC2s do not acquire type 1 features, such as interferon (IFN)-γ production or T-bet expression and do not respond to IL-12 stimulation. Instead, STAT4 is activated by type I IFNs and supports the maintenance of the iILC2 pool. Transcriptomic analysis of Stat4-deficient ILC2s reveals enhanced type I IFN signaling and impaired proliferation, suggesting that STAT4 functions to antagonize IFN-driven suppression. Our data uncover a novel regulatory axis in which IL-25-induced STAT4 expression equips ILC2s to modulate interferon responses and to prevent aberrant autocrine function of type I IFNs, thus sustaining inflammatory effector populations during immune activation. These findings broaden the understanding of ILC2 activation and suggest new avenues for modulating innate lymphocytes in inflammatory diseases.

Gliomas constitute one of the most aggressive and heterogeneous forms of brain tumors, posing major challenges for understanding their biology and developing effective treatments. Animal models enable the collection of rich longitudinal datasets describing tumor dynamics, which can be integrated within mathematical models to elucidate the biological mechanisms governing tumor growth. While most formulations rely on reaction–diffusion systems with limited insight on tissue deformation and fluid transport, we propose a magnetic resonance imaging (MRI)-informed, poroelastic model to describe C6 glioma growth in rats. We use data from animals (n=4) that were imaged five times after intracranial injection of cancer cells. Each MRI dataset includes (i) anatomical T1-weighted data for brain and tumor segmentation and to assign mechanical properties; (ii) diffusion-weighted MRI, which enables estimation of the fraction of each voxel that is tumor; and (iii) dynamic contrast-enhanced MRI, which informs permeability as well as vascular and liquid fraction maps. Using finite-element simulations, model calibration for each rat uses the Gauss–Newton method informed by the first three MRI datasets. Tumor forecasts are validated by assessing model-data agreement on the remaining two MRI datasets. Our results show relative tumor volume errors between 0.94% and 11.27% at calibration, and prediction errors between 4.73% and 36.03%. Additionally, Dice scores ranged from 0.80 to 0.93 during calibration, and from 0.75 to 0.93 during validation. Thus, our results suggest that our poromechanical model can describe C6 glioma growth. This study provides a first step towards a patient-specific, multiscale model of the spatiotemporal poromechanics underlying glioma progression and therapeutic response.

Figure S1. Neutrophil deficiency is associated with increased lymphoid aggregates. Related to Figure 1.A) Number of total lymphoid structures and B) number of lymphoid structures with follicular dendritic cells detected by RNAscope staining for the follicular dendritic cell marker Mfge8 in colon tissue sections from Csf3r+/+(n=8) and Csf3r-/- (n=5) mice after AOM/DSS-induced CRC. C) Representative images of RNAscope analysis for Mfge8 in colon tissue sections from Csf3r-/- mice after AOM/DSS-induced CRC: aggregates with follicular structures are visible as Mfge8+ (red spot): on the top is visible a lymphoid aggregate at two different magnifications, with diffuse Mfge8+ cells; on the bottom right lymphoid microaggregate with Mfge8+ cells; on the bottom left aggregate without follicular structures (Mfge8+). D) Representative dot-plot of neutrophils frequency in blood (top panels) and colon LP (bottom panels) of Csf3r-/- mice and Csf3r-/- mice 4hrs and 18hrs after adoptive transfer of neutrophils. E-F) Frequency of neutrophils in blood (D) and colon LP (E) of DSS-treated Csf3r-/- mice (n=5) and Csf3r-/- mice 4hrs (n=5) and 18hrs (n=5) after adoptive transfer of neutrophils. A-B) Representative data of three independent experiments. C-E) One experiment. A-B, D-E) Unpaired Student’s t-Test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Chemokines and their receptors play a pivotal role in the initiation and regulation of inflammation through the orchestration of leukocyte extravasation and directed migration toward sites of tissue injury. Tight control of chemokine gradients within tissues is essential to ensure that inflammatory responses remain transient and properly resolved. When this regulatory mechanism fails, dysregulated chemokine signaling can contribute to the development of chronic inflammation. Atypical receptors for chemoattractants comprise atypical chemokine receptors (ACKRs) and the chemerin-presenting receptor CCRL2. ACKRs perform specialized functions enabling the fine-tuning of chemokine gradients, primarily through the scavenging, sequestration, or redistribution of chemokines. By regulating their spatial and temporal availability, ACKRs play a key role in limiting excessive leukocyte recruitment and promoting inflammation resolution. The lungs are in a dynamic equilibrium between immune activation and homeostasis. Rapid and tightly regulated immune cell recruitment is essential for effective host defense while preventing tissue damage. In this context, ACKRs expressed by specialized lung endothelial cells are emerging as critical regulators of leukocyte trafficking and inflammatory resolution. Given the paucity of studies in this area, this review summarizes current knowledge of ACKRs and CCRL2 in lung immune surveillance and discusses their potential as therapeutic targets in lung diseases.

Supplementary Table 2. List of critical reagents and antibodies used.

Figure S6. Neutrophil infiltration is associated with tissue repair gene signatures in patients with ulcerative colitis. Related to Figure 6. A-E) Enrichment plots for gene ontology term epithelial cell (GO:Epithelial Cell Proliferation (A, D); GO:Epithelial Cell Development (B, E); GO:Maintanance of Gastrointestinal Epithilium (C)) in CSF3Rhigh versus CSF3Rlow UC patients GSE109142 (A-C) GSE87473 (D-E). F-K) Enrichment plots for gene ontology term antimicrobial response (GO:Response to molecule of bacterial origin (F, I); GO:Defense response to bacterium; GO:Antimicrobial humoral immune response mediated by antimicrobial peptides) in CSF3Rhigh versus CSF3Rlow UC patients GSE109142 (F-H) and GSE87473 (I-K). CSF3R gene expression values were stratified by quartiles and patients belonging to the upper (GSE109142 n=51; GSE87473 n=21) and lower GSE109142 n=51; GSE97473 n=21) quartiles were considered for differential expression analysis. FDR, false discovery rate.

Natural killer (NK) cells are central to innate antitumor immunity, yet their function is systemically compromised in colorectal cancer (CRC). We show that CRC patients exhibit early and pronounced alterations in peripheral blood NK cells, marked by a reduced frequency of total CD56⁺ cells, a shift toward the CD56lowCD16⁺ subset, and impaired cytotoxic and cytokine responses. Notably, plasma from CRC patients, particularly those with advanced disease, induces similar dysfunctions in healthy donor (HD) NK cells, suppressing mTORC1 signalling and effector activity. Transcriptomic profiling of HD NK cells exposed to CRC plasma revealed downregulation of TNF-α signalling components, concordant with reduced systemic and intracellular TNF-α levels in patients. Additionally, CRC plasma suppressed the JAK-STAT pathway and upregulated SOCS family genes, further dampening NK cell responsiveness. Inhibition of TNF-α in HD NK cells recapitulated the CRC plasma-induced defects, while exogenous TNF-α partially restored NK cell function, including STAT5 and S6 phosphorylation. These findings uncover TNF-α signalling deficiency as a systemic mechanism of NK cell suppression in CRC, linking impaired metabolism to immune evasion. Targeting this axis may offer a novel strategy to reinvigorate NK cell-mediated antitumor immunity in CRC.

Colorectal cancer (CRC) treatment represents a major clinical challenge, with immunotherapy providing durable responses only in a minority of patients. A deeper understanding of CD8⁺ T cell exhaustion and its contribution to immune checkpoint inhibitor (ICI) responsiveness is essential for the development of more effective therapeutic strategies. Preclinical models that faithfully reproduce the immune landscape of human CRC are therefore critical to address these challenges. Here, we established a syngeneic organoid-based orthotopic CRC mouse model by transplanting quadruple mutant Apc⁻/⁻KrasG12D/+Trp53R172H/⁻Smad4⁻/⁻ (AKPS) intestinal organoids into the rectal submucosa of immunocompetent mice. Single-cell transcriptomic profiling revealed that CD8⁺ T cells represent the predominant leukocyte population within the tumor infiltrate and comprise populations transitioning toward dysfunction. Functionally, CD8⁺ T cell depletion led to increased tumor burden in orthotopic AKPS implants, underscoring their antitumor activity. Importantly, anti-PD-1 treatment increased the abundance of dysfunctional CD8⁺ T cell populations within AKPS tumors and reduced tumor growth, demonstrating the responsiveness of this model to ICIs. In contrast, subcutaneous implants of AKPS were infiltrated by mixed CD4⁺ and CD8⁺ T cell subsets, with CD8⁺ T cells exhibiting a markedly less dysfunctional profile, highlighting the limitations of heterotopic tumor models for studying antitumor immune responses. Together, our findings establish the AKPS orthotopic CRC model as a platform to dissect the molecular mechanisms of early CD8⁺ T cell dysfunction and to preclinically evaluate novel immunotherapeutic interventions in CRC.

BACKGROUND:Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide and is characterized by an immunosuppressive tumor microenvironment (TME). While adaptive immunity contributes to tumor control, growing evidence underscores the role of innate lymphocytes, particularly natural killer (NK) cells, in early antitumor surveillance. However, tumor-infiltrating NK cells often exhibit defective maturation and impaired effector functions, whereas the signals regulating NK cell differentiation and activity in CRC remain poorly defined. Interleukin-27 (IL-27) has emerged as a regulator of antitumor immunity, yet its role in modulating NK cell responses in intestinal tumors is largely unexplored. METHODS:We employed an orthotopic transplantation model of genetically engineered colorectal tumor organoids Apc-/-KrasG12D/+Trp53R172H/-Smad4-/- (AKPS) to investigate NK cell heterogeneity, maturation, and function during CRC progression. Tumor-infiltrating innate lymphocytes were analyzed using single-cell RNA sequencing, flow cytometry, immunofluorescence, and functional assays. In vitro co-culture systems and in vivo IL-27 blockade were used to assess the impact of tumor-derived IL-27 on NK cell transcriptional programs and effector activity. RESULTS:Single-cell transcriptomic profiling revealed marked heterogeneity among tumor-infiltrating NK cells, identifying subsets with different maturation states and functional capacities. Among innate lymphocytes, AKPS tumors were dominated by NK cells displaying reduced activating receptors and diminished cytotoxic and cytokine-producing potential. Phenotypic and adoptive transfer analyses demonstrated that the TME favors persistence of immature CD27+ NK cell subsets while limiting differentiated NK cells. Mechanistically, tumor-derived IL-27 emerged as a critical regulator sustaining NK cell infiltration and activation; IL-27 blockade reduced NK cell accumulation, interferon-γ production, and immature subset frequency. The clinical relevance of these findings was supported by analysis of human CRC single-cell datasets, which revealed elevated IL-27 signaling in intratumoral NK cells, as well as by the ability of patient-derived organoids to enhance NK-cell cytotoxicity, which was reduced by IL-27 blockade. CONCLUSIONS:Our study identifies IL-27 as a critical modulator of NK cell differentiation and function in CRC, highlighting its role in sustaining NK cell-mediated immune surveillance within the tumor microenvironment. These findings provide mechanistic insight into NK cell dysfunction in CRC and suggest IL-27 signaling as a promising therapeutic target to restore innate antitumor immunity.

Figure S4. Neutrophil deficiency is associated with decreased expression of IL-22 by γδ T cells. Related to Figure 4-5. A-C) Representative gating strategy used to gate myeloid population (A), lymphoid population (B) and innate lymphoid cells (ILC) (C) in colon LP. UTC: unconventional T cells (TCRβ+, CD8-, CD4-). D) IL-22 expression in myeloid cells derived from DSS-treated Csf3r+/+(n=3), Csf3r-/-(n=3) stimulated 4 hours with IL-23 analyzed by FACS. E-F) Expression of IL-22 by lymphoid cells derived from DSS-treated Csf3r+/+(n=4), Csf3r-/- (n=4) stimulated 4 hours with IL-23 plus IL-1β (E) and PMA plus ionomycin (F) analyzed by FACS. A-F) Representative data of three independent experiments. D-F) Multiple t-test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.