Development of immune and microbial environments is independently regulated in the mammary gland.

Breastfeeding is important for mammals, providing immunological and microbiological advantages to neonates, together with the nutritional supply from the mother. However, the mechanisms of this functional diversity in the mammary gland remain poorly characterized. Here, we show that, similar to the gastrointestinal tract, the mammary gland develops immune and microbial environments consisting of immunoglobulin A (IgA) and the microflora, respectively, both of which are important for protecting neonates and the mother from infectious diseases. The IgA production and microflora development are coordinated in the gastrointestinal tract but seem to be independently regulated in the mammary gland. In particular, the chemokine (C-C motif) ligand 28 and poly-Ig receptor, crucial molecules for the IgA production in milk, were expressed normally in germ-free lactating mice but were almost undetectable in postweaning mothers, regardless of the microflora presence. Our findings offer insights into potentially improving the quality of breastfeeding, using both immunological and microbiological approaches.

Nanogel-based nasal ghrelin vaccine prevents obesity.

Obesity is associated with multiple comorbidities such as cardiovascular diseases and has a huge economic impact on the health-care system. However, the treatment of obesity remains insufficient in terms of efficacy, tolerability, and safety. Here we created a nasal vaccine against obesity for the first time. To avoid the injectable administration-caused pain and skin-related adverse event, we focused on the intranasal route of antigen delivery. We developed a vaccine antigen (ghrelin-PspA (pneumococcal surface protein A)), which is a recombinant fusion protein incorporating ghrelin, a hormone that stimulates food intake and decreases energy expenditure, and PspA, a candidate of pneumococcal vaccine as a carrier protein. Ghrelin-PspA antigen was mixed with cyclic di-GMP adjuvant to enhance the immunogenicity and incorporated within a nanometer-sized hydrogel for the effective antigen delivery. Intranasal immunization with ghrelin-PspA vaccine elicited serum immunoglobulin G antibodies against ghrelin and attenuated body weight gain in diet-induced obesity mice. This obesity-attenuating effect was caused by a decrease in fat accumulation and an increase in energy expenditure that was partially due to an increase in the expression of mitochondrial uncoupling protein 1 in brown adipose tissue. The development of this nasal vaccine provides a new strategy for the prevention and treatment of obesity.

Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c+ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC-B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP-TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal infection.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer's patch M cells.

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell-specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B-deficient mice showed reduced expression of most, but not all, other M-cell-specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B-deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B-deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

Implications of nasopharynx-associated lymphoid tissue (NALT) in the development of allergic responses in an allergic rhinitis mouse model.

BACKGROUND: Nasopharynx-associated lymphoid tissue (NALT) serves as an important inductive site for mucosal immunity in the upper respiratory tract. Despite its importance in the mucosal immune system, little is known regarding the role of NALT in airway allergic immune responses. We aimed to elucidate the role of NALT in the induction of upper airway allergic responses in a mouse model. METHODS: Inhibitor of DNA binding/differentiation 2 (Id2)(-/-) and Id2(+/-) mice was exposed to the ovalbumin (OVA)-induced allergic rhinitis model, because the former resulted in the NALT deficiency. The allergic parameters, such as allergic symptoms, serum OVA-specific immunoglobulin E (IgE) levels, eosinophil infiltration, and cytokine profiles in the nasal mucosa, were compared between Id2(-/-) and Id2(+/-) groups. RESULTS: NALT-null, Id2(-/-) mice displayed significantly lower allergic responses compared with Id2(+/-) mice, as demonstrated by lower levels of allergic symptoms, serum OVA-specific IgE, eosinophilic infiltration, and local Th2 cytokine transcriptions. To determine which of two factors, that is, the absence of NALT or the alteration of immunocompetent cell populations caused by the Id2 deficiency, has a larger effect on the attenuated allergic immune responses in Id2(-/-) mice, lethally irradiated Id2(-/-) mice were engrafted with C57BL/6 wild-type bone marrow cells and showed still significantly lower allergic immune responses compared with equally treated Id2(+/-) mice. In addition, IgE class switch recombination-associated molecules, such as ε immunoglobulin heavy-chain germline gene transcript, ε mRNA, and activation-induced cytidine deaminase mRNA, were detected in NALT from OVA-sensitized wild-type mice. CONCLUSION: These results show the critical role of NALT for the induction of allergic responses in the upper airway at least in part by means of class switching to IgE in situ.

M cell-targeted mucosal vaccine strategies.

Immune responses in the aerodigestive tract are characterized by production and transport of specific IgA antibodies across the epithelium to act as a first line of defense against pathogens in the external environment. To sample antigens on mucosal surfaces in the intestine and upper respiratory tract, the immune system relies on a close collaboration between specialized antigen-sampling epithelial M cells and lymphoid cells. Depending on various factors, local antigen presentation in the mucosal tissue leads to tolerance or initiation of an active immune response. Recently, molecules that could be used to target vaccine antigens to apical M cell surfaces have been identified. Here we review the M cell-targeted vaccine strategy, an approach that could be used to enhance uptake and efficacy of vaccines delivered in the nasal cavity or intestine.

Terminology: nomenclature of mucosa-associated lymphoid tissue.

Stimulation of mucosal immunity has great potential in vaccinology and immunotherapy. However, the mucosal immune system is more complex than the systemic counterpart, both in terms of anatomy (inductive and effector tissues) and effectors (cells and molecules). Therefore, immunologists entering this field need a precise terminology as a crucial means of communication. Abbreviations for mucosal immune-function molecules related to the secretory immunoglobulin A system were defined by the Society for Mucosal Immunolgy Nomenclature Committee in 1997, and are briefly recapitulated in this article. In addition, we recommend and justify standard nomenclature and abbreviations for discrete mucosal immune-cell compartments, belonging to, and beyond, mucosa-associated lymphoid tissue.

A marvel of mucosal T cells and secretory antibodies for the creation of first lines of defense.

The mucosal immune system acts as a first line of defense against bacterial and viral infections while also playing a crucial role in the establishment and maintenance of mucosal homeostasis between the host and the outside environment. In addition to epithelial cells and antigen-presenting cells (dendritic cells and macrophages), B and T lymphocytes form a dynamic mucosal network for the induction and regulation of secretory IgA (S-IgA) and cytotoxic T lymphocyte (CTL) responses. This review seeks to shed light on the pathways of induction and regulation of these responses and to elucidate the role they simultaneously play in fending off pathogen invasion and maintaining mucosal homeostasis.

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Development of antigen induced colitis in SCID mice reconstituted with spleen derived memory type CD4(+) CD45RB(+) T cells.

BACKGROUND AND AIMS: Enteric bacterial and/or food antigens may be crucial in the development of colitis but little is known of the exact mechanism of antigen specific reactions in this condition. The aim of this study was to determine whether systemically primed antigen specific CD4(+) T cells containing both CD45RB(high) and CD45RB(low) populations participate as a pathogenic subset that in turn leads to inflammatory reactions selectively in the large intestine. METHODS: SCID mice were reconstituted with splenic CD4(+) CD45RB(+) T cells or CD4(+) CD45RB(low) T cells isolated from donor mice systemically primed with ovalbumin (OVA) plus CFA. The reconstituted mice were then fed OVA for several weeks. RESULTS: Reconstitution of SCID mice with OVA primed splenic CD4(+) T cells, containing populations of CD45RB(high) and CD45RB(low), resulted in the development of colitis by 4-5 weeks following repeated administration of oral OVA. Histopathological study revealed thickened wall, inflammatory cell infiltration, crypt elongation, and loss of goblet cells in the large intestine. The CD4(+) CD45RB(low) population of cells extracted from the affected large intestine secreted high levels of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) at the protein and mRNA levels. Administration of neutralising antibodies to TNF-alpha, but not to IFN-gamma, prevented the development of colitis. Furthermore, adoptive transfer with OVA primed splenic CD4(+) CD45RB(low) T cells evoked severe colitis. CONCLUSIONS: These results demonstrate that systemically primed activated/memory CD4(+) CD45RB(low) T cells can mediate the development of specific antigen induced colitis in SCID mice, and also that TNF-alpha is critical in the induction of this type of colitis. Our results contrast with those from studies in some colitis models in which CD45RB(low) T cells appeared to prevent colitis through secretion of immunosuppressive cytokines.

Highly purified mutant E112K of cholera toxin elicits protective lung mucosal immunity to diphtheria toxin.

We demonstrated that the mutant of cholera toxin (mCT) E112K which was LPS-free supported the induction of protective immunity in mucosal (e.g. lung lavage) and systemic (e.g. serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice. Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K or native CT (nCT) was co-administered nasally with DT. The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands. Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT. The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4+ T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT. The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective. Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity. These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g. lung lavages and the lungs).

HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient conditions.

In the vaccine strategy against HIV, bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of potential vectors for mucosal delivery of vaccine Ag. We analyzed the induction of the Ag-specific Ab response by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that can secrete the V3 principal neutralizing epitope of HIV. Mice were nasally immunized with rBCG-V3J1 (10 microg) three times at weekly intervals. Four weeks after the initial immunization, high titers of V3J1-specific IgG Abs were seen in serum. These high levels of HIV-specific serum IgG responses were maintained for >12 mo following nasal immunization without any booster immunization. V3J1-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinated mice. Nasal rBCG-V3J1 also induced high levels of prolonged HIV-specific serum IgG responses in Th1 (IFN-gamma(-/-))- or Th2 (IL-4(-/-))-immunodeficient mice. Further, IgG3 was highest among V3 peptide-specific IgG subclass Ab responses in these immunodeficient mice as well as in wild-type mice. In addition, this Ag-specific serum IgG Abs induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize clinical isolate of HIV in vitro. These results suggested that the nasal rBCG-V3J1 system might be used as a therapeutic vaccine in addition to a prophylaxis vaccine for the control of AIDS.

Influence of enterotoxin on mucosal intranet: selective inhibition of extrathymic T cell development in intestinal intraepithelial lymphocytes by oral exposure to heat-labile toxin.

We tested the possibility that heat-labile enterotoxin of Escherichia coli (LT) affects the development of extrathymic T cells in the intraepithelial lymphocyte (IEL) compartment. After oral administration of LT, the number of extrathymic CD8alphaalpha+ IEL was selectively and significantly diminished when compared with the corresponding cells in phosphate-buffered saline-fed control mice. To clarify the mechanism behind this selective reduction of CD8alphaalpha+ IEL, we analyzed the expression of essential cytokines and their corresponding receptors for the mucosal intranet formed by intestinal epithelial cells (IEC) and IEL. The expression levels of stem cell factor, interleukin (IL)-7, and IL-15 in IEC, and their corresponding receptors, i. e. c-kit, IL-7 receptor, and IL-15 receptor, in CD8alphaalpha+ IEL were reduced following oral feeding with LT. These findings suggest that LT negatively regulates development of CD8alphaalpha+ IEL via the disruption of mucosal intranet-associated cytokine and cytokine receptors, which are required for the development and/or expansion of extrathymically developed T cells. Further, LT-induced destruction of the mucosal intranet resulted in the impairment of IEC generation via an increase of apoptosis.

The pulmonary environment promotes Th2 cell responses after nasal-pulmonary immunization with antigen alone, but Th1 responses are induced during instances of intense immune stimulation.

The purpose of this study was to determine the nature of the CD4(+) Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4(+), and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-gamma, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-gamma) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4(+) Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1beta expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.

A revisit of mucosal IgA immunity and oral tolerance.

Induction of mucosal immunity by oral immunization with protein antigen alone is difficult: potent mucosal adjuvants, vectors, or other special delivery systems are required. Cholera toxin (CT) has been shown to be an effective adjuvant for the development of mucosal vaccines and, when given with vaccine, induces both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, and in addition to potential type-I hypersensitivity, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its inherent toxicity. When we examined the potential toxicity of CT for the central nervous system, both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development. Tolerance represents the most common and important response of the host to environmental antigens, including food and commensal bacterial components, for the maintenance of an appropriate immunological homeostasis. We have examined whether Peyer patches could play a more important role for the maintenance of oral tolerance. Using Peyer patch-null mice, we found that mice lacking this gut-associated lymphoid tissue retained their capability to produce secretory IgA antibodies but did not develop normal oral tolerance to protein antigens.

Nasal administration of cholera toxin (CT) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE).

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, elicits mucosal adjuvant activities by inducing antigen-specific CD4+ T cells secreting T helper type 2 (Th2) cytokines. Experimental autoimmune encephalomyelitis (EAE) is induced by Th1 cells specific for myelin-derived antigens. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55 and CT was nasally administered as an immunomodulator on day 7 following MOG challenge. Clinical severity in the CT-treated mice was milder when compared to PBS-treated mice, while the levels of expression of interleukin (IL)-12 and interferon (IFN)-gamma in the central nervous system (CNS) of CT-treated mice were lower than PBS-treated mice. Thus, nasal administration of the mucosal immunomodulator CT ameliorated the severity of EAE, which was associated with the suppression of Th1 cell responses.