Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo, with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker-treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow haematopoietic stem cell population. Importantly, although the haematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3-blocker-treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the haematopoietic system.

Diverse myeloid cells are recruited to the developing and inflamed mammary gland.

The immune system plays fundamental roles in the mammary gland, shaping developmental processes and controlling inflammation during infection and cancer.Here, we reveal unanticipated heterogeneity in the myeloid cell compartment duringdevelopment of virgin, pregnant, lactating and involuting mouse mammary glands,and in milk. We investigate the functional consequences of individual and compoundchemokine receptor deficiency on cell recruitment. Diverse myeloid cell recruitmentwas also shown in models of sterile inflammation and bacterial infection.Strikingly, we have shown that inflammation and infection can alter the abundanceof terminal end buds, a key developmental structure, within the pubertal mammarygland. This previously unknown effect of inflammatory burden during puberty couldhave important implications for understanding pubertal development.

Chemokine receptors coordinately regulate macrophage dynamics and mammary gland development.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We have previously demonstrated that the atypical chemokine receptor ACKR2 contributes to the control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor that mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here, we reveal reciprocal regulation of branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition, oestrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets.

Regnase-1 degradation is crucial for IL-33- and IL-25-mediated ILC2 activation.

Group 2 innate lymphoid cells (ILC2s) are a critical innate source of type 2 cytokines in allergic inflammation. Although ILC2s are recognized as a critical cell population in the allergic inflammation, the regulatory mechanism(s) of ILC2s are less well understood. Here, we show that Regnase-1, an immune regulatory RNAse that degrades inflammatory mRNAs, negatively regulates ILC2 function and that IκB kinase (IKK) complex-mediated Regnase-1 degradation is essential for IL-33- and IL-25-induced ILC2 activation. ILC2s from Regnase-1AA/AA mice expressing a Regnase-1 S435A/S439A mutant resistant to IKK complex-mediated degradation accumulated Regnase-1 protein in response to IL-33 and IL-25. IL-33- and IL-25-stimulated Regnase-1AA/AA ILC2s showed reduced cell proliferation and type 2 cytokine (IL-5, IL-9, and IL-13) production and increased cell death. In addition, Il2ra and Il1rl1, but not Il5, Il9, or Il13, mRNAs were destabilized in IL-33-stimulated Regnase-1AA/AA ILC2s. In vivo, Regnase-1AA/AA mice showed attenuated acute type 2 pulmonary inflammation induced by the instillation of IL-33, IL-25, or papain. Furthermore, the expulsion of Nippostrongylus brasiliensis was significantly delayed in Regnase-1AA/AA mice. These results demonstrate that IKK complex-mediated Regnase-1 degradation is essential for ILC2-mediated type 2 responses both in vitro and in vivo. Therefore, controlling Regnase-1 degradation is a potential therapeutic target for ILC2-contributed allergic disorders.

Placental chemokine compartmentalisation: A novel mammalian molecular control mechanism.

Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2-/-embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as 'intermediate' cells. CC chemokine receptor 2 (CCR2)-/-embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2-/-embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences.

Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis.

BACKGROUND: Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). OBJECTIVE: We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. METHODS: We performed an in vitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. A human nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption in vitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier in vivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. RESULTS: rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption in vitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption in vivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. CONCLUSION: Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.

Barrier dysfunction in the nasal allergy.

Epithelial cells form the first physiological barrier against invasion by pathogens and the infiltration of allergens. Tight junctions (TJ), a cell-cell junctional complex located on the apical side of epithelial cells, have a critical role in the maintenance of epithelial barrier function. Impaired TJ structures are observed in patients with asthma, atopic dermatitis and nasal allergy; therefore, the dysfunction of epithelial barriers might be involved in the initiation or progression of allergic diseases. Protease-containing allergens and environmental pollutants enhance paracellular transport in epithelial cells through disruption of epithelial barrier function. This suggests that the disruption of TJ leads to the promotion of allergen delivery into the subepithelia, resulting in the progression of allergic diseases. Thus, protection of the epithelial barrier function might prevent or inhibit the development or exacerbation of allergic diseases. Recently, we reported that diesel exhaust particles (DEP), the main component of particulate patter 2.5, exacerbated allergic rhinitis (AR) in a mouse model through TJ disruption. In addition, we revealed that the oxidative stress-mediated pathway is involved in the effects caused by DEP and that nasal treatment with a reactive oxygen species (ROS) scavenger suppressed DEP-induced TJ disruption and exacerbation of AR. In this review, we focus on the relationship between TJ disruption and allergic disease. Furthermore, we discuss our recent findings regarding TJ disruption and the exacerbation of AR.

Activation of group 2 innate lymphoid cells exacerbates and confers corticosteroid resistance to mouse nasal type 2 inflammation.

Both Th2 cells and group 2 innate lymphoid cells (ILC2s) contribute to allergic diseases. However, their exact role and relationship in nasal allergic disorders are unclear. In this study, we investigated the cooperation of Th2 cells and ILC2s in a mouse model of nasal allergic disorder. To differentially activate Th2 cells and/or ILC2s in nasal mucosa, mice were intra-nasally administered ovalbumin (OVA) antigen, papain, an ILC2-activator, or both for 2 weeks. Epithelial thickness and number of eosinophils in the nasal mucosa were evaluated at 24 h after the final challenge. Intra-nasal administration of OVA and papain preferentially activated Th2 cells and ILC2s, respectively, in the nose. Both OVA and papain increased the nasal epithelial thickness and number of eosinophils, and their coadministration significantly enhanced the symptoms. Although T-/B-cell-deficient mice showed severely decreased nasal symptoms induced by OVA or OVA-plus-papain, the mice still showed slight papain-induced nasal symptoms. In ILC2-deficient mice, OVA-plus-papain-induced nasal symptoms were suppressed to the same level as OVA-alone. Similarly, IL-33- and ST2-deficient mice showed decreased OVA-plus-papain-induced nasal symptoms. IL-5 induced eosinophilia only, but IL-13 contributed to both nasal epithelial thickening and eosinophilia induced by OVA-plus-papain. Dexamethasone ameliorated OVA-alone-induced nasal epithelial thickening. However, OVA-plus-papain-induced nasal epithelial thickening was only partially controlled by dexamethasone. These results demonstrate that IL-33/ST2-pathway-mediated ILC2 activation exacerbated Th2-cell-induced nasal inflammation by producing IL-13. Although Th2-cell-alone-induced nasal inflammation was controlled by corticosteroid treatment, the activation of ILC2s conferred treatment resistance. Therefore, ILC2s and their activators could be therapeutic targets for treatment-refractory nasal allergic disorders.

Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice.

BACKGROUND: Allergen-mediated cross-linking of IgE on mast cells/basophils is a well-recognized trigger for type 1 allergic diseases such as allergic rhinitis (AR). However, allergens may not be the sole trigger for AR, and several allergic-like reactions are induced by non-IgE-mediated mechanisms. OBJECTIVE: We sought to describe a novel non-IgE-mediated, endotoxin-triggered nasal type-1-hypersensitivity-like reaction in mice. METHODS: To investigate whether endotoxin affects sneezing responses, mice were intraperitoneally immunized with ovalbumin (OVA), then nasally challenged with endotoxin-free or endotoxin-containing OVA. To investigate the role of T cells and mechanisms of the endotoxin-induced response, mice were adoptively transferred with in vitro-differentiated OVA-specific TH2 cells, then nasally challenged with endotoxin-free or endotoxin-containing OVA. RESULTS: Endotoxin-containing, but not endotoxin-free, OVA elicited sneezing responses in mice independent from IgE-mediated signaling. OVA-specific TH2 cell adoptive transfer to mice demonstrated that local activation of antigen-specific TH2 cells was required for the response. The Toll-like receptor 4-myeloid differentiation factor 88 signaling pathway was indispensable for endotoxin-containing OVA-elicited rhinitis. In addition, LPS directly triggered sneezing responses in OVA-specific TH2-transferred and nasally endotoxin-free OVA-primed mice. Although antihistamines suppressed sneezing responses, mast-cell/basophil-depleted mice had normal sneezing responses to endotoxin-containing OVA. Clodronate treatment abrogated endotoxin-containing OVA-elicited rhinitis, suggesting the involvement of monocytes/macrophages in this response. CONCLUSIONS: Antigen-specific nasal activation of CD4+ T cells followed by endotoxin exposure induces mast cell/basophil-independent histamine release in the nose that elicits sneezing responses. Thus, environmental or nasal residential bacteria may exacerbate AR symptoms. In addition, this novel phenomenon might explain currently unknown mechanisms in allergic(-like) disorders.

Murine allergic rhinitis and nasal Th2 activation are mediated via TSLP- and IL-33-signaling pathways.

Thymic stromal lymphopoietin (TSLP) and IL-33 are epithelium-derived proallergic cytokines that contribute to allergic diseases. Although the involvement of TSLP in allergic rhinitis (AR) is suggested, the exact role of TSLP in AR is poorly understood. Furthermore, the relative contribution of TSLP and IL-33 in nasal allergic responses has not been described. In this study, we examined the roles of TSLP and IL-33 in AR by analyzing acute and chronic AR models. Acute AR mice were intraperitoneally immunized with ragweed, then intranasally challenged with ragweed pollen for four consecutive days. Chronic AR mice were nasally administrated ragweed pollen on consecutive days for 3 weeks. In both models, TSLP receptor (TSLPR)-deficient mice showed defective sneezing responses and reduced serum ragweed-specific IgE levels compared with wild-type (WT) mice. Analyses of bone-marrow chimeric mice demonstrated that hematopoietic cells were responsible for defective sneezing in TSLPR-deficient mice. In addition, FcεRI(+)-cell-specific TSLPR-deficient mice showed partial but significant reduction in sneezing responses. Of note, Th2 activation and nasal eosinophilia were comparable between WT and TSLPR-deficient mice. ST2- and IL-33-deficient mice showed defective Th2 activation and nasal eosinophilia to acute, but not chronic, ragweed exposure. TSLPR and ST2 double-deficient mice showed defective Th2 activation and nasal eosinophilia even after chronic ragweed exposure. These results demonstrate that TSLPR signaling is critical for the early phase response of AR by controlling the IgE-mast-cell/basophil pathway. The IL-33/ST2 pathway is central to nasal Th2 activation during acute allergen exposure, but both TSLPR and ST2 contribute to Th2 responses in chronically allergen-exposed mice.

The role of basophils and proallergic cytokines, TSLP and IL-33, in cutaneously sensitized food allergy.

Cutaneous sensitization with a food antigen before its consumption elicits the development of food allergy. Here, we report the site- and stage-dependent roles of basophils and proallergic cytokines, thymic stromal lymphopoietin (TSLP) and IL-33, in a mouse model of food allergy initially sensitized cutaneously with the food antigen. Mice were epicutaneously sensitized with the food antigen ovalbumin (OVA) followed by oral challenge with OVA. Epicutaneously sensitized mice produced OVA-specific IgE and developed IgE-dependent anaphylaxis after oral challenge. Basophil-depleted or TSLP-receptor-deficient mice did not produce OVA-specific IgE and were protected from oral challenge-induced anaphylaxis. IL-33-deficient mice produced normal levels of OVA-specific IgE. However, IL-33-deficient mice and mice treated with recombinant soluble IL-33 receptor were protected from anaphylaxis. Thus, basophils and TSLP have pivotal roles in Th2 development in the skin during the sensitization phase of food allergy. In contrast, while IL-33 is dispensable for promoting cutaneous antigen sensitization, the cytokine is essential for inducing IgE-dependent anaphylaxis in the gut.

Pathogenic Th2-type follicular helper T cells contribute to the development of lupus in Fas-deficient mice.

Fas mutant mice are well recognized as autoimmune mouse models, which develop symptoms similar to human systemic lupus erythematosus. Although disease severity in Fas mutant mice is greatly affected by the genetic background, the mechanisms affecting pathological heterogeneity among different strains of Fas mutant mice are poorly understood. In this study, we examined the phenotypic differences between Fas-deficient (Fas (-/-)) mice on the BALB/c and C57BL/6 backgrounds to gain insight into the etiological and pathological heterogeneity of monogenic autoimmune diseases. Fas (-/-) mice on the BALB/c background (BALB/c-Fas (-/-)) developed more severe autoimmune disease with high serum auto-antibodies and renal disease compared with those on the C57BL/6 background (C57BL/6-Fas (-/-)). Splenic B cells were highly activated, and germinal center formation was enhanced in BALB/c-Fas (-/-) but not in C57BL/6-Fas (-/-) mice. Follicular helper T (Tfh) cells were equally abundant in the spleens from both strains of Fas (-/-) mice. However, Tfh cells from BALB/c-Fas (-/-) mice produced much higher amounts of B-cell-activating cytokines, including IL-4 and IL-10, a phenotype reminiscent of Th2-type Tfh cells described in human studies. Our results revealed a qualitative difference in Tfh cells between the two strains of Fas (-/-) mice. We propose that the pathogenic Th2-type Tfh cells in BALB/c-Fas (-/-) mice contribute to the excessive activation of B cells, resulting in high serum immunoglobulin levels and the severe lupus phenotype, which may account for the differential outcomes of human monogenic autoimmune diseases.

Identification of a novel type 2 innate immunocyte with the ability to enhance IgE production.

Fas (CD95), a member of the tumor necrosis factor receptor superfamily, mediates apoptosis-inducing signals in its expressing cells, especially in self-reactive cells. We recently reported that Fas(-/-) mice with a BALB/c background (BALB/c Fas(-/-) mice) developed blepharitis with allergic inflammation that was accompanied by hyper-IgE production. Here, we found a novel type of immunocyte in the spleen of BALB/c Fas(-/-) mice, which enhanced the production of IgE by B cells in the presence of IL-4 and CD40 signaling in vitro. The immunocyte did not express lineage markers but expressed Thy-1 and Sca-1 just like recently identified type 2 innate lymphoid cells, such as natural helper (NH) cells and nuocytes. However, they did not express c-Kit, IL-7R and IL-33R (T1/ST2), important markers of type 2 innate lymphoid cells. Instead, our identified Lin(-)Thy-1(+)Sca-1(+) cells expressed IL-18R and secreted Th2 cytokines when co-cultured with B cells or when stimulated with IL-18 and IL-2. Moreover, we found essentially the same type of cells in BALB/c wild-type mice as in BALB/c Fas(-/-) mice, which enhanced IgE production in contact with B cells in vitro. These cells from BALB/c wild-type mice expressed Fas and were sensitive to Fas-mediated apoptosis. Collectively, the newly identified Lin(-)Thy-1(+)Sca-1(+) cell, which we designated a F-NH cell (Fas-expressing natural helper cell), is a novel type 2 innate immunocyte with activity to enhance IgE production from B cells with the help of IL-4 and CD40 signaling. F-NH cells may play an important role in the development of chronic allergic inflammation.

Fas deficiency in mice with the Balb/c background induces blepharitis with allergic inflammation and hyper-IgE production in conjunction with severe autoimmune disease.

Fas (CD95) is a cell surface death receptor belonging to the tumor necrosis factor receptor superfamily, which mediates apoptosis-inducing signaling when activated by Fas ligand or its agonistic antibody. lpr mice with a loss of apoptosis-inducing function mutation in the Fas gene develop systemic autoimmune disease and lymphadenopathy but not allergic inflammation. In the case of Fas mutations including lpr and knockout (KO), background genes determine the incidence and severity of lymphadenopathy and histopathological manifestation of systemic autoimmunity: MRL-lpr/lpr mice and C57BL/6-lpr/lpr or C57BL/6 Fas KO mice develop severe and minimum disease, respectively. We generated Fas KO mice with the Balb/c background that show severer autoimmune phenotypes than MRL-lpr/lpr mice, such as critical infiltration of mononuclear cells into lung, liver and spleen, elevated serum levels of auto-antibodies and a decreased life span. To our astonishment, Balb/c Fas KO mice spontaneously develop blepharitis with not only autoimmune inflammation with deposition of auto-antibody but also allergic inflammation with infiltration by eosinophils and mast cells and show the capacity to strongly increase serum level of IgE and IgG1 along with their aging. Thus, Fas expression regulates development of not only autoimmune disease but also allergic inflammation.

Dclk1 distinguishes between tumor and normal stem cells in the intestine.

There is great interest in tumor stem cells (TSCs) as potential therapeutic targets; however, cancer therapies targeting TSCs are limited. A drawback is that TSC markers are often shared by normal stem cells (NSCs); thus, therapies that target these markers may cause severe injury to normal tissues. To identify a potential TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was reported as a candidate NSC marker in the gut, but recent reports have implicated it as a marker of differentiated cells (for example, Tuft cells). Using lineage-tracing experiments, we show here that Dclk1 does not mark NSCs in the intestine but instead marks TSCs that continuously produce tumor progeny in the polyps of Apc(Min/+) mice. Specific ablation of Dclk1-positive TSCs resulted in a marked regression of polyps without apparent damage to the normal intestine. Our data suggest the potential for developing a therapy for colorectal cancer based on targeting Dclk1-positive TSCs.

Malignant NK/T-cell lymphoma associated with simian Epstein-Barr virus infection in a Japanese macaque (Macaca fuscata).

A case of spontaneous malignant lymphoma in a Japanese macaque (Macaca fuscata) was pathologically, etiologically and virologically studied. Nasal cavity was involved in the neoplastic lesions in addition to lymphoid and visceral tissues. Histopathological analyses revealed the presence of neoplastic cells classified into histiocytic Hodgkin-like cells and Reed-Sternberg-like cells. Histiocytic Hodgkin-like cells were CD16+ and CD20+, and the CD16+ cells were also positive for simian Epstein-Barr virus (sEBV)-encoded early RNA transcripts. RS-like cells were negative for CD3, CD16 and CD20. Antibodies to early antigen of sEBV were detected, while antibodies to simian T-cell leukemia virus-1 were negative. The case may correspond to EBV-associated nasal type NK/T-cell lymphoma in humans rather than Hodgkin lymphoma.

Immunohistochemical detection of beta-amyloid and beta-amyloid precursor protein in the canine brain and non-neuronal epithelial tissues.

We examined the immunohistochemical localization of beta-amyloid (Abeta) and beta-amyloid precursor protein (AFPP) in the neuronal and non-neuronal tissues of 14 aged (over 10 years old) and 4 young (0-4 years old) dogs. Abeta was detected only in the senile plaque in the cerebrum of aged dogs. AbetaPP was expressed in the neuronal cell body, neuronal fiber, senile plaque and perimalacic tissue independent of age. In addition, epithelial cells in the bronchus, bronchial glands, gastric and intestinal mucosa, intrahepatic bile ducts, and pancreatic ducts and exocrine glands were immunopositive for AbetaPP. Thus, it is suggested that AbetaPP may be expressed in the non-neuronal epithelial tissues independent of age and of the presence of senile plaques, and the expression may depend on individual differences or physiological conditions.

Thymoma in a dog with a part of granular cell proliferation and concurrent lymphoma cells.

A 12-year-old male Shiba dog showed anemia and the swelling of systemic lymph nodes. X-ray and post mortal examinations revealed a anterior mediastinal mass. Histologically, the tumor mass consisted of four different elements; cord-like proliferation of cuboidal epithelial cells, tubular or cystic structures lined with ciliated epithelial cells, proliferation of large round-shaped epithelial cells with PAS-slightly positive granular cytoplasm, and diffuse proliferation of neoplastic lymphocytes. Epithelial cells in cord-like or cystic structures were strongly positive for cytokeratin. Granular or foamy cells were negative for all markers examined and had myelin-like bodies in the cytoplasm by electron microscopy. The neoplastic lymphocytes in the tumor mass were considered being derived from concurrent multicentric lymphoma. Based on these findings, the present case was diagnosed as thymoma with a part of granular cell proliferation and concurrent lymphoma cells.