InVivoMAb anti-mouse Ly6G/Ly6C (Gr-1)

Ischemic stroke results in blood-brain barrier (BBB) disruption, during which the reciprocal interaction between ischemic neurons and components of the BBB appears to play a critical role. However, the underlying mechanisms for BBB protection remain largely unknown. In this study, we found that Serpina3n, a serine protease inhibitor, was significantly upregulated in the ischemic brain, predominantly in ischemic neurons from 6 hours to 3 days after stroke. Using neuron-specific adeno-associated virus (AAV), intranasal delivery of recombinant protein, and immune-deficient Rag1-/- mice, we demonstrated that Serpina3n attenuated BBB disruption and immune cell infiltration following stroke by inhibiting the activity of granzyme B (GZMB) and neutrophil elastase (NE) secreted by T cells and neutrophils. Furthermore, we found that intranasal delivery of rSerpina3n significantly attenuated the neurologic deficits after stroke. In conclusion, Serpina3n is a novel ischemic neuron-derived proteinase inhibitor that counterbalances BBB disruption induced by peripheral T cell and neutrophil infiltration after ischemic stroke. These findings reveal a novel endogenous protective mechanism against BBB damage with Serpina3n being a potential therapeutic target in ischemic stroke.

The Consensus Molecular Subtype 4 (CMS4) of colorectal cancer (CRC) has the worst prognosis and the highest frequency of hepatic metastases. It is characterized by abundant cancer-associated fibroblasts (CAFs) in the tumor microenvironment and active TGFβ signaling, but the molecular drivers of metastasis remain unclear. Here, we show that TGFβ signaling in CRC patient-derived CAFs from the primary tumor induces production of IL-6 family cytokines, particularly IL-6 and IL-11. These cytokines stimulate hepatocytes to express myeloid chemoattractants, including SAA1, through gp130-dependent JAK/STAT signaling. This promotes neutrophil recruitment to the liver, potentially creating a pro-metastatic niche. This IL-6 family-JAK/STAT stromal signaling axis is active in both a murine model of CMS4 as well as in patients with human CRC in vivo. Combined, our data reveal that TGFβ-driven CAF signaling actively contributes to the formation of a neutrophil-dependent, pre-metastatic hepatic niche in the metastatic phenotype of CMS4 CRC.

The immunosuppressive tumor microenvironment (TME) drives radioresistance, but the role of γδ T cells in regulating radiosensitivity remains incompletely understood. In this study, we found that γδ T cell infiltration in the TME substantially increased after radiotherapy and contributed to radioresistance. Depletion of γδ T cells enhanced radiosensitivity. Single-cell RNA sequencing revealed that γδ T cells in the post-radiotherapy TME were characterized by the expression of Zbtb16, Il23r, and Il17a, and served as the primary source of IL-17A. These γδ T cells promoted radioresistance by recruiting myeloid-derived suppressor cells and suppressing T cell activation. Mechanistically, radiotherapy-induced tumor cell-derived microparticles containing dsDNA activated the cGAS-STING/NF-κB signaling pathway in macrophages, upregulating the expression of the chemokine CCL20, which was critical for γδ T cell recruitment. Targeting γδ T cells and IL-17A enhanced radiosensitivity and improved the efficacy of radiotherapy combined with anti-PD-1 immunotherapy, providing potential therapeutic strategies to overcome radioresistance.

Inflammatory bowel diseases (IBDs) constitute chronic inflammatory disease of the gastrointestinal tract, with escalating global prevalence. There is a pressing demand for safe and effective treatments for IBDs. Fibroblast growth factor 1 (FGF1) variant FGF1ΔHBS, characterised by reduced mitogenic capacity, has shown promising therapeutic potential in various inflammatory conditions, including obesity and diabetic nephropathy. Hence, exploring the therapeutic impact of FGF1ΔHBS on colitis is warranted.

The diversity of neutrophils' subtypes and functions in the tumor microenvironmentunderlies their contributions to both pro- and anti-tumorigenic activities. How to elucidate the antitumor mechanisms of neutrophil and effectively utilize it still needs to be studied. In this study, neutrophil stimulus reaction index (NSRI) was established for high selectivity of neutrophils. Sufficient and highly selective exogenous neutrophils were used to infuse into tumor-bearing mice. Navigation technique was used to make neutrophils rapidly infiltrate into the tumor tissue, which played a key role in inhibiting tumor growth. We found that tumor cells were efficiently killed in direct coculture with sufficient neutrophils by promoting a multimodal death primarily dominated by apoptosis. Interestingly, colon cancer organoid diameters were decreased markedly cocultured with neutrophils, and were more remarkable in the application of organoid microinjection of neutrophils. Consistent with the 3D-printed model, in vivo model indicated that neutrophils meaningfully inhibited the tumor growth by formation of neutrophil extracellular traps (NETs) with neutrophil elastase (NE) payload. This study not only unveils the crucial anticancer effects of neutrophils but also sets out an innovative strategy of aggressive dose exogenous neutrophils with precise navigation, which provided the basis for advancements in neutrophil-based immunotherapy for colon cancer treatment.

Inflammation is increasingly recognized as a risk factor for psychiatric disorders. Animal models of stress and stress-related disorders are associated with blood neutrophilia. The mechanistic relevance of this to symptoms or behavior is unclear. We characterized the immune response to chronic social defeat (CSD) stress at brain border regions in male mice. Here we show that chronic, but not acute, stress causes neutrophil accumulation in the meninges-i.e., "meningeal neutrophilia"- but not the brain. CSD promotes neutrophil trafficking to meninges via vascular channels originating from skull bone marrow (BM). Transcriptional analysis suggests CSD increases type I interferon (IFN-I) signaling in meningeal neutrophils. Blocking this pathway via the IFN-I receptor (IFNAR) protects against the negative behavioral effects of CSD stress. Our identification of IFN-I signaling as a putative mediator of meningeal neutrophil recruitment may facilitlate development of new therapies for stress-related disorders.

The literature suggests that hepatocellular Yes-associated protein 1 (YAP1) signaling is activated following hepatectomy and that such activation can suppress the growth of metastatic liver tumors. The prognosis of a real-world cohort of 240 patients with colorectal cancer liver metastasis (CRLM) undergoing major and minor hepatectomy was compared after adjusting for confounding factors. To model CRLM, we induced liver metastasis in mice by transsplenically injecting MC38 cells. We found that patients with CRLM and mice undergoing major hepatectomy had better survival compared to those undergoing minor hepatectomy. Mechanistically, extensive hepatectomy activates hepatocellular YAP1 by regulating the epidermal growth factor receptor, altering glutamine metabolism-related gene expression and increasing liver glutamine consumption. This metabolic shift leads to glutamine scarcity in tumor cells, causing increased reactive oxygen species production, which promotes loss of YAP1 activity in tumor cells. Consequently, the production of the chemokine CXCL5 is suppressed, which inhibits myeloid-derived suppressor cell infiltration and enhancing the immunological function of CD8+ T cells.

Current treatments for ischemic stroke focus on removing neurovascular-occluding clots but overlook the resulting neuroinflammation. Clinical trials targeting brain-infiltrating peripheral immune cells have failed to improved outcomes, leaving mechanisms of neuroinflammation poorly understood. Whilst many studies have examined the inflammatory cells and cytokine profile of the post-stroke brain at days and weeks following injury onset, we propose that interventions at these timepoints are too late to limit brain damage. In this study, we examined brain immune cell composition, cytokine levels, and neurological dysfunction at hyperacute (3 h) and acute (24 h) stages following a preclinical mouse model of ischemic stroke. Interestingly, we detected elevated inflammatory cytokines in brain tissue as early as 3 h, notably before infiltrating neutrophil and monocyte arrival at 24 h. Depletion of peripheral immune cells by antibodies or genetic alteration did not dampen neuroinflammation, nor improve sensorimotor function. Intravital imaging of Cx3cr1gfp/+ mice showed that microglia, the brain-resident immune cell, display rapidly altered morphology, and swiftly upregulated cytokine production hyperacutely post-stroke. Altogether, our study provides evidence that microglia are key drivers of early neuroinflammation and modulating their function at clinically-relevant timepoints will improve stroke recovery.

Many metastatic clear cell renal cell carcinomas (ccRCC) are resistant to immune checkpoint inhibitor therapies, however the mechanisms underlying sensitivity or resistance remain incompletely characterised. We demonstrate that ccRCCs in the Vhl/Trp53/Rb1 mutant mouse model are resistant to combined anti-PD-1/anti-CTLA-4 therapy alone and in combination with additional therapeutic agents that reflect current ccRCC clinical trials. However, in some animals in vivo checkpoint therapy allowed isolated splenic T cells to recognise cultured ccRCC cells from the same animal, implicating the tumour microenvironment in suppression of T cell activation. We identified putative immunosuppressive myeloid cell populations with features similar to myeloid cells in the microenvironment of human ccRCC. The expression patterns of immune checkpoint ligands in both the mouse model and in human ccRCC suggests that several checkpoint systems other than PD-1 and CTLA-4 are likely to represent the dominant T cell suppressive forces in ccRCC. Our findings characterise an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance and pave the way for a systematic functional dissection of the identified potential molecular barriers to effective immune therapy of ccRCC.

Sterile acute kidney injury (AKI) is common in the clinic and frequently associated with unexplained hypoxemia that does not improve with dialysis. AKI induces remote lung inflammation with neutrophil recruitment in mice and humans, but which cellular cues establish neutrophilic inflammation and how it contributes to hypoxemia is not known. Here we report that AKI induced rapid intravascular neutrophil retention in lung alveolar capillaries without extravasation into tissue or alveoli, causing hypoxemia by reducing lung capillary blood flow in the absence of substantial lung interstitial or alveolar edema. In contrast to direct ischemic lung injury, lung neutrophil recruitment during remote lung inflammation did not require cues from intravascular nonclassical monocytes or tissue-resident alveolar macrophages. Instead, lung neutrophil retention depended on the neutrophil chemoattractant CXCL2 released by activated classical monocytes. Comparative single-cell RNA-Seq analysis of direct and remote lung inflammation revealed that alveolar macrophages were highly activated and produced CXCL2 only in direct lung inflammation. Establishing a CXCL2 gradient into the alveolus by intratracheal CXCL2 administration during AKI-induced remote lung inflammation enabled neutrophils to extravasate. We thus discovered important differences in lung neutrophil recruitment in direct versus remote lung inflammation and identified lung capillary neutrophil retention that negatively affected oxygenation by causing a ventilation-perfusion mismatch as a driver of AKI-induced hypoxemia.

Integrin Mac-1 plays a critical role in the development of multiple sclerosis (MS); however, the underlying mechanism is not fully understood. Here, we developed a myeloid-specific Mac-1-deficient mouse. Using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we report that Mac-1 on myeloid cells is key to disease development. Our data reveal that myeloid-specific Mac-1 significantly increases EAE severity and hinders disease regression. Loss of Mac-1 increases Gr-1+ cells in peripheral tissues and the CNS and preferably accelerates the transition of Ly6Chi monocytes from a pro-inflammatory to an immunosuppressive phenotype in a disease stage-dependent manner. Mechanistically, our results demonstrate that Mac-1 suppresses interferon-γ production and prevents monocytes from acquiring immunosuppressive functions by reducing the expression of iNOS, IDO, and CD84. Administration of a NOS-specific inhibitor in Mac-1-deficient EAE mice abolishes disease regression. These insights could help develop Mac-1-targeting strategies for better treatment of MS.

Cigarette smoke (CS) exposure amplifies neutrophil accumulation. IL-35, a novel cytokine with anti-inflammatory properties, is involved in protection against asthma. However, the biological roles of neutrophils and the precise molecular mechanisms of IL-35 in CS exposed-asthma remain unclear. We showed that the exacerbation of CS exposed-asthma leads to dramatically increased neutrophil counts and an imbalance in DC-Th17/Treg immune responses. RNA sequencing revealed that NETs, part of a key biological process in neutrophils, were significantly upregulated in the context of CS exposed-asthma exacerbation and that IL-35 treatment downregulated NET-associated gene expression. Targeted degradation of NETs, rather than neutrophil depletion, alleviated the CS exposed-asthma. Mechanistically, STAT3 phosphorylation promoted ferroptosis, exacerbating NET release, which in turn enhanced dendritic cell (DC) antigen presentation, activated T cells, and specifically promoted Th17 cell differentiation while inhibiting Treg cells. IL-35 acting on the gp130 receptor alleviated STAT3-mediated ferroptosis-associated NET formation. In summary, our study revealed a novel mechanism by which IL-35 inhibited NET formation, subsequently alleviating neutrophilic inflammation and restoring the DC-Th17/Treg imbalance in CS exposed-asthma, highlighting the potential of IL-35 as a targeted therapeutic strategy.

Bladder cancer (BLCA) is a challenging malignancy with a poor prognosis, particularly in muscle-invasive cases. Despite recent advancements in immunotherapy, response rates remain suboptimal. This study investigates the role of METTL3, an m6A RNA methylation "writer," in regulating the immune microenvironment of BLCA.

Disease tolerance is a host response to infection that limits collateral damage to host tissues while having a neutral effect on pathogen fitness. Previously, we found that the pathogenic lactic acid bacterium Streptococcus pyogenes manipulates disease tolerance using its aerobic mixed-acid fermentation pathway via the enzyme pyruvate dehydrogenase, but the microbe-derived molecules that mediate communication with the host's disease tolerance pathways remain elusive. Here we show in a murine model that aerobic mixed-acid fermentation inhibits the accumulation of inflammatory cells including neutrophils and macrophages, reduces the immunosuppressive cytokine interleukin-10, and delays bacterial clearance and wound healing. In infected macrophages, the aerobic mixed-acid fermentation end-products acetate and formate from streptococcal upregulate host acetyl-CoA metabolism and reduce interleukin-10 expression. Inhibiting aerobic mixed-acid fermentation using a bacterial-specific pyruvate dehydrogenase inhibitor reduces tissue damage during murine infection, correlating with increased interleukin-10 expression. Our results thus suggest that reprogramming carbon flow provides a therapeutic strategy to mitigate tissue damage during infection.

Fulminant myocarditis (FM) is a severe inflammatory condition of the myocardium that often results in sudden death, particularly in young individuals. In this study, we employed single-nucleus and spatial transcriptomics to perform a comprehensive analysis of coxsackievirus B3 (CVB3)-induced FM in A/J mice, spanning seven distinct time points pre- and post-treatment. Our findings reveal that mesothelial cells play a critical role in the early stage of myocarditis by acting as primary targets for CVB3 infection. This triggers the activation of macrophages, initiating a cascade of inflammation. Subsequently, pro-inflammatory Inflammatory_Mac and T cells infiltrate the myocardium, driving tissue damage. We also identified Cd8+ effector T cells as key mediators of cardiomyocyte injury. These cells release cytotoxic molecules, particularly IFN-γ, which modulates the expression of Spi1, a factor implicated in exacerbating cardiomyocyte death and amplifying disease progression. Therapeutic interventions targeting the IFN-γ/Spi1 axis demonstrated significant efficacy in FM models. Notably, intravenous immunoglobulin (IVIG) treatment reduced mortality, suppressed viral proliferation, and mitigated the hyperinflammatory state of FM. IVIG therapy also downregulated IFN-γ and Spi1 expression, underscoring its immunomodulatory and therapeutic potential. This comprehensive spatiotemporal transcriptomic analysis provides profound insights into the pathogenesis of FM and highlights actionable therapeutic targets, paving the way for more effective management strategies for this life-threatening condition.

Although immune checkpoint blockade (ICB) therapy represents a bright spot in antitumor immunotherapy, its clinical benefits in colorectal cancer (CRC) are limited. Therefore, a new target for mediating CRC immunosuppression is urgently needed. Adenomatous polyposis coli (APC) mutations have been reported as early-stage characteristic events in CRC, but the role of truncated APC in the CRC immune microenvironment remains unclear and its clinical significance has yet to be explored.

Using chromosomal barcoding, we observed that >97% of the Streptococcus pneumoniae (Spn) population turns over in the lung within 2 days post-inoculation in a murine model. This marked collapse of diversity and bacterial turnover was associated with acute inflammation (severe pneumococcal pneumonia), high bacterial numbers in the lungs, bacteremia, and mortality. Intra-strain competition mediated by the blp locus, which expresses bacteriocins in a quorum-sensing-dependent manner, was required for each of these effects. Bacterial turnover from the activity of Blp-bacteriocins increased the release of the pneumococcal toxin, pneumolysin (Ply), which was sufficient to account for the lung pathology. The ability of Ply to evade complement, rather than its pore-forming activity, prevented opsonophagocytic clearance of Spn enabling its multiplication in the lung, facilitating the inflammatory response and subsequent invasion into the bloodstream. Thus, our study demonstrates how an appreciation for bacterial population dynamics during infection provides new insight into pathogenesis.

Ubiquitin regulatory X (UBX) domain-containing protein 6 (UBXN6) is an essential cofactor for the activity of the valosin-containing protein p97, an adenosine triphosphatase associated with diverse cellular activities. Nonetheless, its role in cells of the innate immune system remains largely unexplored. In this study, we report that UBXN6 is upregulated in humans with sepsis and may serve as a pivotal regulator of inflammatory responses via the activation of autophagy. Notably, the upregulation of UBXN6 in sepsis patients was negatively correlated with inflammatory gene profiles but positively correlated with the expression of Forkhead box O3, an autophagy-driving transcription factor. Compared with those of control mice, the macrophages of mice subjected to myeloid cell-specific UBXN6 depletion exhibited exacerbated inflammation, increased mitochondrial oxidative stress, and greater impairment of autophagy and endoplasmic reticulum-associated degradation pathways. UBXN6-deficient macrophages also exhibited immunometabolic remodeling, characterized by a shift to aerobic glycolysis and elevated levels of branched-chain amino acids. These metabolic shifts amplify mammalian target of rapamycin pathway signaling, in turn reducing the nuclear translocation of the transcription factor EB and impairing lysosomal biogenesis. Together, these data reveal that UBXN6 serves as an activator of autophagy and regulates inflammation to maintain immune system suppression during human sepsis.

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb-/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4+ T cells and most predominantly CD8+ T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb-/- and IL-10-/- mice. Furthermore, resistance against OMP was restored in Tcrb-/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4+ and CD8+ T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma.