Maternal Lactobacillus rhamnosus administration impacts neonatal CD4 T-cell activation and prevents murine T helper 2-type allergic airways disease.

Gut microbiota plays a role in the neonatal immune education and could influence susceptibility to Th2-type immune disorders, such as allergies, the most prevalent chronic diseases in early childhood. We studied the impact of oral Lactobacillus rhamnosus (L.rhamnosus) supplementation to pregnant/breastfeeding C57BL/6 mice on the development of allergic airways disease in their offspring. We observed that mice, from L.rhamnosus-treated mothers, inoculated with ovalbumin (OVA)-Aluminium hydroxide (ALUM) at 3 days of life and challenged intranasally 4 weeks later showed decreased Th2-associated cytokines, IgE and IgG1, lung eosinophilia and airway hyper-reactivity compared to OVA-sensitized mice from untreated mothers. In that setting, the L.rhamnosus treatment increased the number and maturation of splenic neonatal type 1 conventional dendritic cells (cDC1) that remained largely dominant over the cDC2 and favored their OVA-specific Th1 differentiation. In response to inhaled house dust mite (HDM) allergen, the maternal L.rhamnosus supplementation increased the number of neonatal pulmonary cDC1 expressing lower amount of costimulatory molecules compared with no supplementation and decreased the number of cDC2 without affecting their costimulatory molecules expression. An HDM-specific Foxp3+RORγt+ Treg polarization was monitored in the lung draining lymph nodes. Finally, we confirmed the inhibitory effect of maternal L.rhamnosus treatment on all the measured features of the HDM allergic airways reaction in their offspring. We conclude that maternal L.rhamnosus administration prevents Th2-type allergic airways disease in their neonates by favoring splenic cDC1/Th1 responses against ALUM-adjuvanted OVA or by promoting a pulmonary Foxp3+RORγt+ Treg activation against inhaled HDM.

Very early-life exposure to microbiota-induced TNF drives the maturation of neonatal pre-cDC1.

OBJECTIVE: Induction of immune protection against pathogens is particularly crucial during the neonatal period dominated by anti-inflammatory and tolerance immunity. The preclinical study was carried out to determine whether environmental factors such as microbiota may influence early life immunity by impacting the development and the functional maturation of precursors of type 1 conventional dendritic cells (pre-cDC1), endowed with regulatory properties. DESIGN: Pre-cDC1 phenotype and cytokine expression in the spleen of neonates from antibiotic-treated mothers were established. The role of myeloid-derived tumour necrosis factor (TNF) was tested in vitro and in vivo. RNA sequencing analysis on neonatal sorted pre-cDC1 was performed. The early life protective CD8+ T-cell response against Listeria monocytogenes was monitored. RESULTS: We observed that first exposure to microbiota promotes TNF secretion by monocytes and macrophages shortly after birth. We demonstrated that this myeloid-derived inflammatory cytokine is crucial to induce the maturation of these neonatal regulatory pre-cDC1. Myeloid TNF signalling acts on C1q and ß-catenin pathway and modifies the fatty acid metabolism in neonatal pre-cDC1. Furthermore, we showed that during neonatal L. monocytogenes infection, microbiota-associated myeloid TNF promotes the capacity of these pre-cDC1 to induce protective CD8+ T-cell responses, by modulating their ability to secrete interleukin-10 (IL-10) and IL-12p40. CONCLUSION: Our findings emphasise the role of microbiota-derived TNF to kick-start the differentiation and the functional maturation of the neonatal splenic pre-cDC1 compartment. They bring a better understanding of potential mechanisms underlying some microbiota-linked immune dysfunction in early life.

Myeloid tumor necrosis factor and heme oxygenase-1 regulate the progression of colorectal liver metastases during hepatic ischemia-reperfusion.

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-ß, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity.

Hepatocarcinoma Induces a Tumor Necrosis Factor-Dependent Kupffer Cell Death Pathway That Favors Its Proliferation Upon Partial Hepatectomy.

Partial hepatectomy (PH) is the main treatment for early-stage hepatocellular carcinoma (HCC). Yet, a significant number of patients undergo recursion of the disease that could be linked to the fate of innate immune cells during the liver regeneration process. In this study, using a murine model, we investigated the impact of PH on HCC development by bioluminescence imaging and flow cytometry. While non-resected mice were able to control and reject orthotopic implanted Hepa1-6 hepatocarcinoma cells, resected liver underwent an increased tumoral proliferation. This phenomenon was associated with a PH-induced reduction in the number of liver-resident macrophages, i.e., Kupffer cells (KC). Using a conditional ablation model, KC were proved to participate in Hepa1-6 rejection. We demonstrated that in the absence of Hepa1-6, PH-induced KC number reduction was dependent on tumor necrosis factor-alpha (TNF-α), receptor-interacting protein kinase (RIPK) 3, and caspase-8 activation, whereas interleukin (IL)-6 acted as a KC pro-survival signal. In mice with previous Hepa1-6 encounter, the KC reduction switched toward a TNF-α-RIPK3-caspase-1 activation. Moreover, KC disappearance associated with caspase-1 activity induced the recruitment of monocyte-derived cells that are beneficial for tumor growth, while caspase-8-dependent reduction did not. In conclusion, our study highlights the importance of the TNF-α-dependent death pathway induced in liver macrophages following partial hepatectomy in regulating the antitumoral immune responses.

HO-1 mitigates acute kidney injury and subsequent kidney-lung cross-talk.

Ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), which contributes to the development of chronic kidney disease (CKD). IRI-induced AKI releases proinflammatory cytokines (e.g. IL-1ß, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in proinflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur. Acute lung injury (ALI) is a major remote organ dysfunction associated with AKI. Hence, kidney-lung cross-talk remains a clinical challenge, especially in critically ill population. The stress-responsive enzyme, heme oxygenase-1 (HO-1) is largely known to protect against renal IRI and may be preventively induced using hemin prior to renal insult. However, the use of hemin-induced HO-1 to prevent AKI-induced ALI remains poorly investigated. Mice received an intraperitoneal injection of hemin or sterile saline 1 day prior to surgery. Twenty-four hours later, mice underwent bilateral renal IRI for 26 min or sham surgery. After 4 or 24 h of reperfusion, mice were sacrificed. Hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation, and oxidative stress). This protective effect was associated with a dampened systemic inflammation (i.e. IL-6 and KC). Subsequently, mitigated lung inflammation was found in hemin-treated mice (i.e. neutrophils influx and lung KC). The present study demonstrates that hemin-induced HO-1 controls the magnitude of renal IRI and the subsequent AKI-induced ALI. Therefore, targeting HO-1 represents a promising approach to prevent the impact of renal IRI on distant organs, such as lung.

Dual effect of hemin on renal ischemia-reperfusion injury.

Acute kidney injury (AKI) is a major public health concern, which is contributing to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia-reperfusion injury (IRI) remains a leading cause of AKI. The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known to be effective. Therefore, HO-1 might be an interesting therapeutic target in case of predictable AKI (e.g. partial nephrectomy or renal transplantation). However, the use of hemin to mitigate established AKI remains poorly characterized. Mice underwent bilateral renal IRI for 26 min or sham surgery. After surgical procedure, animals were injected either with hemin (5 mg/kg) or vehicle. Twenty-four hours later, mice were sacrificed. Despite strong HO-1 induction, hemin-treated mice exhibited significant renal damage and oxidative stress as compared to vehicle-treated mice. Interestingly, higher dose of hemin is associated with more severe IRI-induced AKI in a dose-dependent relation. To determine whether hemin preconditioning remains efficient to dampen postoperative hemin-amplified IRI-induced AKI, we pretreated mice either with hemin (5 mg/kg) or vehicle 24 h prior to surgical procedure. Then, all mice (hemin- and vehicle-pretreated) received postoperative injection of hemin (5 mg/kg) to amplify IRI-induced AKI. In comparison to vehicle, prior administration of hemin to renal IRI mitigated hemin-amplified IRI-induced AKI as attested by fewer renal damage, inflammation and oxidative stress. In conclusion, hemin may have a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.

Specific expression of heme oxygenase-1 by myeloid cells modulates renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is a major risk factor for delayed graft function in renal transplantation. Compelling evidence exists that the stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against IRI. However, the role of myeloid HO-1 during IRI remains poorly characterized. Mice with myeloid-restricted deletion of HO-1 (HO-1M-KO), littermate (LT), and wild-type (WT) mice were subjected to renal IRI or sham procedures and sacrificed after 24 hours or 7 days. In comparison to LT, HO-1M-KO exhibited significant renal histological damage, pro-inflammatory responses and oxidative stress 24 hours after reperfusion. HO-1M-KO mice also displayed impaired tubular repair and increased renal fibrosis 7 days after IRI. In WT mice, HO-1 induction with hemin specifically upregulated HO-1 within the CD11b+ F4/80lo subset of the renal myeloid cells. Prior administration of hemin to renal IRI was associated with significant increase of the renal HO-1+ CD11b+ F4/80lo myeloid cells in comparison to control mice. In contrast, this hemin-mediated protection was abolished in HO-1M-KO mice. In conclusion, myeloid HO-1 appears as a critical protective pathway against renal IRI and could be an interesting therapeutic target in renal transplantation.

IL-12p40/IL-10 Producing preCD8α/Clec9A+ Dendritic Cells Are Induced in Neonates upon Listeria monocytogenes Infection.

Infection by Listeria monocytogenes (Lm) causes serious sepsis and meningitis leading to mortality in neonates. This work explored the ability of CD11c(high) lineage DCs to induce CD8+ T-cell immune protection against Lm in mice before 7 days of life, a period symbolized by the absence of murine IL-12p70-producing CD11c(high)CD8α+ dendritic cells (DCs). We characterized a dominant functional Batf3-dependent precursor of CD11c(high) DCs that is Clec9A+CD205+CD24+ but CD8α- at 3 days of life. After Lm-OVA infection, these pre-DCs that cross-present Ag display the unique ability to produce high levels of IL-12p40 (not IL-12p70 nor IL-23), which enhances OVA-specific CD8+ T cell response, and regulatory IL-10 that limits OVA-specific CD8+ T cell response. Targeting these neonatal pre-DCs for the first time with a single treatment of anti-Clec9A-OVA antibody in combination with a DC activating agent such as poly(I:C) increased the protection against later exposure to the Lm-OVA strain. Poly(I:C) was shown to induce IL-12p40 production, but not IL-10 by neonatal pre-DCs. In conclusion, we identified a new biologically active precursor of Clec9A+ CD8α- DCs, endowed with regulatory properties in early life that represents a valuable target to augment memory responses to vaccines.

The cholinergic anti-inflammatory pathway delays TLR-induced skin allograft rejection in mice: cholinergic pathway modulates alloreactivity.

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.

Neonatal follicular Th cell responses are impaired and modulated by IL-4.

Newborns are characterized by poor responses to vaccines. Defective B cell responses and a Th2-type polarization can account for this impaired protection in early life. We in this study investigated the generation of follicular Th (TFH) cells, involved in the development of Ab response and germinal center reaction, upon vaccination in neonates. We showed that, compared with adults, Ab production, affinity maturation, and germinal center formation were reduced in neonates immunized with OVA-aluminum hydroxide. Although this vaccination induced CD4(+) CXCR5(+) PD-1(+) TFH cells in newborns, their frequency, as well as their Bcl6 expression and IL-21 and IL-4 mRNA induction, was decreased in early life. Moreover, neonatal TFH cells were mainly localized in interfollicular regions of lymphoid tissues. The prototypic Th2 cytokine IL-4 was found to promote the emergence and the localization in germinal centers of neonatal TFH cells, as well as the neonatal germinal center reaction itself. In addition, IL-4 dampened expression of Th17-related molecules in neonatal TFH cells, as TFH cells from immunized IL-4-deficient neonates displayed enhanced expression of RORγt and IL-17. This Th17-like profile correlated with an increased secretion of OVA-specific IgG2a. Our study thus suggests that defective humoral immunity in early life is associated with limited and IL-4-modulated TFH cell responses.

Interferon regulatory factor 3 deficiency leads to interleukin-17-mediated liver ischemia-reperfusion injury.

UNLABELLED: Interferon regulatory factor 3 (IRF3) is an important transcription factor in Toll-like receptor 4 (TLR4) signaling, a pathway that is known to play a critical role in liver ischemia-reperfusion injury. In order to decipher the involvement of IRF3 in this setting, we first compared the intensity of hepatic lesions in IRF3-deficient versus wildtype mice. We found increased levels of blood transaminases, enhanced liver necrosis, and more pronounced neutrophil infiltrates in IRF3-deficient mice. Neutrophil depletion by administration of anti-Ly6G monoclonal antibody indicated that neutrophils play a dominant role in the development of severe liver necrosis in IRF3-deficient mice. Quantification of cytokine genes expression revealed increased liver expression of interleukin (IL)-12/IL-23p40, IL-23p19 messenger RNA (mRNA), and IL-17A mRNA in IRF3-deficient versus wildtype (WT) mice, whereas IL-27p28 mRNA expression was diminished in the absence of IRF3. The increased IL-17 production in IRF3-deficient mice was functionally relevant, as IL-17 neutralization prevented the enhanced hepatocellular damages and liver inflammation in these animals. Evidence for enhanced production of IL-23 and decreased accumulation of IL-27 cytokine in M1 type macrophage from IRF3-deficient mice was also observed after treatment with lipopolysaccharide, a setting in which liver gamma-delta T cells and invariant natural killer T cells were found to be involved in IL-17A hyperproduction. CONCLUSION: IRF3-dependent events downstream of TLR4 control the IL-23/IL-17 axis in the liver and this regulatory role of IRF3 is relevant to liver ischemia-reperfusion injury.

The p110δ isoform of the kinase PI(3)K controls the subcellular compartmentalization of TLR4 signaling and protects from endotoxic shock.

Lipopolysaccharide activates plasma-membrane signaling and endosomal signaling by Toll-like receptor 4 (TLR4) through the TIRAP-MyD88 and TRAM-TRIF adaptor complexes, respectively, but it is unclear how the signaling switch between these cell compartments is coordinated. In dendritic cells, we found that the p110δ isoform of phosphatidylinositol-3-OH kinase (PI(3)K) induced internalization of TLR4 and dissociation of TIRAP from the plasma membrane, followed by calpain-mediated degradation of TIRAP. Accordingly, inactivation of p110δ prolonged TIRAP-mediated signaling from the plasma membrane, which augmented proinflammatory cytokine production while decreasing TRAM-dependent endosomal signaling that generated anti-inflammatory cytokines (interleukin 10 and interferon-ß). In line with that altered signaling output, p110δ-deficient mice showed enhanced endotoxin-induced death. Thus, by controlling the 'topology' of TLR4 signaling complexes, p110δ balances overall homeostasis in the TLR4 pathway.

Regulation of Th2 responses and allergic inflammation through bystander activation of CD8+ T lymphocytes in early life.

Th2-biased immune responses characterizing neonates may influence the later onset of allergic disease. The contribution of regulatory T cell populations in the prevention of Th2-driven pathologies in early life is poorly documented. We investigated the potential of CD8(+) T cells stimulated at birth with alloantigens to modulate the development of allergic airway inflammation. Newborn mice were immunized with semiallogeneic splenocytes or dendritic cells (DCs) and exposed at the adult stage to OVA aeroallergens. DC-immunized animals displayed a strong Th1 and Tc1/Tc2 alloantigen-specific response and were protected against the development of the allergic reaction with reduced airway hyperresponsiveness, mucus production, eosinophilia, allergen-specific IgE and IgG(1), and reduction of lung IL-4, IL-5, IL-10, and IL-13 mRNA levels. By contrast, splenocyte-immunized mice displayed a Th2 and a weak Tc2 alloantigen-specific response and were more sensitive to the development of the allergen-specific inflammation compared with mice unexposed at birth to alloantigens. DC-immunized animals displayed an important increase in the percentage of IFN-gamma-producing CD8(+)CD44(high), CD8(+)CD62L(high), and CD8(+)CD25(+) subsets. Adoptive transfers of CD8(+) T cells from semiallogeneic DC-immunized animals to adult beta(2)m-deficient animals prevented the development of allergic response, in particular IgE, IL-4, and IL-13 mRNA production in an IFN-gamma-dependent manner, whereas transfers of CD8(+) T cells from semiallogeneic splenocyte-immunized mice intensified the lung IL-4 and IL-10 mRNA level and the allergen-specific IgE. These findings demonstrated that neonatal induction of regulatory CD8(+) T cells was able to modulate key parameters of later allergic sensitization in a bystander manner, without recognition of MHC class I molecules.