Innate IFN-γ Is Essential for Systemic Chlamydia muridarum Control in Mice, While CD4 T Cell-Dependent IFN-γ Production Is Highly Redundant in the Female Reproductive Tract.

Protective immunity against the obligate intracellular bacterium Chlamydia has long been thought to rely on CD4 T cell-dependent gamma interferon (IFN-γ) production. Nevertheless, whether IFN-γ is produced by other cellular sources during Chlamydia infection and how CD4 T cell-dependent and -independent IFN-γ contribute differently to host resistance have not been carefully evaluated. In this study, we dissected the requirements of IFN-γ produced by innate immune cells and CD4 T cells for resolution of Chlamydia muridarum female reproductive tract (FRT) infection. After C. muridarum intravaginal infection, IFN-γ-deficient and T cell-deficient mice exhibited opposite phenotypes for survival and bacterial shedding at the FRT mucosa, demonstrating the distinct requirements for IFN-γ and CD4 T cells in host defense against Chlamydia In Rag1-deficient mice, IFN-γ produced by innate lymphocytes (ILCs) accounted for early bacterial control and prolonged survival in the absence of adaptive immunity. Although type I ILCs are potent IFN-γ producers, we found that mature NK cells and ILC1s were not the sole sources of innate IFN-γ in response to Chlamydia By conducting T cell adoptive transfer, we showed definitively that IFN-γ-deficient CD4 T cells were sufficient for effective bacterial killing in the FRT during the first 21 days of infection and reduced bacterial burden more than 1,000-fold, although mice receiving IFN-γ-deficient CD4 T cells failed to completely eradicate the bacteria from the FRT like their counterparts receiving wild-type (WT) CD4 T cells. Together, our results revealed that innate IFN-γ is essential for preventing systemic Chlamydia dissemination, whereas IFN-γ produced by CD4 T cells is largely redundant at the FRT mucosa.

Autoantigen presentation by splenic dendritic cells is required for RBC-specific autoimmunity.

BACKGROUND: Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. STUDY DESIGN AND METHODS: HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. RESULTS: Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. CONCLUSION: These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

Development, phenotypes of immune cells in BTBR T+Itpr3tf/J mice.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that is characterized by a lack of social interaction, decreased verbal and non-verbal communication skills, and stereotyped repetitive behavior. There is strong evidence that a dysregulated immune response may influence neurodevelopment and thus may have a role in the development of ASD. This study focuses on the characterization of immune cell phenotypes in the BTBR T+Itpr3tf/J (BTBR) mouse strain, a widely used animal model for autism research. Our study demonstrated that BTBR mice have a different immune profile compared to C57BL/6J (B6) mice, which do not display ASD-like characteristics. Thymic cells of BTBR mice have more single positive (SP) CD4+ and CD8+ T cells and fewer double positive (DP) T cells than B6 mice. The development of T cells is increased in BTBR mice with regard to the double negative (DN4) population being much higher in BTBR mice. The spleens and blood of BTBR mice also have more T helper type 1 (Th1), T helper type 2 (Th2) and T regulatory (Treg) cells compared to B6 mice. Aire expression in the thymus and spleen of BTBR mice compared to B6 mice was equivalent and lower, respectively. The mature natural killer (NK) innate immune cell population in blood and spleen is lower in BTBR than B6 mice; NK cell development is blocked prior to the double positive (DN) CD11b+CD27+ stage in BTBR mice. Since BTBR mice have more CD4+ T cells and elevated numbers of Th1 (T-bet+) and Th2 (GATA3+) cells, their low defense against pathogen may be explained by the lower number of NK cells and the significantly lower Th1 to Th2 ratio. The elevated number of plasma cells and autoantibodies of BTBR mice may be due to less presence and function of splenic AIRE.

Comparative Analysis of Bone Marrow-derived Mast Cell Differentiation in C57BL/6 and BALB/c Mice.

Allergic diseases have increased in the last three decades. Mast cells play a critical role in allergic diseases along with allergen-specific immunoglobulin E (IgE). Following mast cell degranulation elicited by ligation of the IgE-FcεRI receptor complex with allergen, allergic reactions are followed by various symptoms such as vascular hyperpermeability, mucous secretion, itching, sneezing, wheezing, rashes, fever, and anaphylactic shock. Susceptibility or inclination to allergy varies depending on individual genetic traits and living environment, and it has long been believed that such an inclination is determined by an immunologic balance of T helper cell types. Mouse strains also have different susceptibilities to allergy. Similar to T helper cells and macrophages, it is not known whether mast cells can also be divided into two different types between mouse strains. In this study, we prepared bone marrow-derived mast cells from BALB/c and C57BL/6 mice and examined their cellular properties. Cellular response to IL-3 and the process of mast cell differentiation from bone marrow cells were different on the basis of cell surface marker molecules. BALB/c-derived cells more efficiently exhibited degranulation than did C57BL/6-derived cells following both calcium ionophore and receptor crosslinking. These functional differences persisted even after a longer cell culture for 8 weeks, suggesting a difference in cell-autonomous characteristics. These results support the concept that mast cells also have different cell types dependent on their genetic background. Abbreviations: Ab: antibody; BMMC: bone marrow-derived mast cell; DNP: dinitrophenyl; FACS: fluorescence-activated cell sorter; FCS: fetal calf serum; FITC: fluorescein isothiocyanate; FSC: forward scatter; HRP: horseradish peroxidase; HSA: human serum albumin; Ig: immunoglobulin; IL: interleukin; MIP-2: macrophage inflammatory protein-2; MCP: mast cell protease; PE: phycoerythrin; PerCP: Peridinin chlorophyll protein complex; SNP: single nucleotide polymorphisms; SSC: side scatter; Th: T helper; TNF-α: tumor necrosis factor alpha.

Genetics and sex influence peripheral and central innate immune responses and blood-brain barrier integrity.

Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. It is appreciated that both humans and mice have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Mouse strain is also an important factor that can contribute to pathophysiological responses to inflammatory stimuli. Therefore, we aimed to test whether BBB disruption and the associated cytokine profiles in response to LPS differed in male and female mice from two mouse strains most commonly used in blood-brain barrier studies: CD-1 and C57BL6/J (C57). Mice were treated with saline, a single injection of 0.3, or 3mg/kg LPS, or three injections of 3mg/kg LPS, and studied 28 hours after the first LPS injection. To assay BBB disruption, we utilized the tracer 99mTc-DTPA. A 23-plex panel of cytokines was assayed in brain and blood of the same cohort of mice, which allowed us to compare differences in the levels of individual cytokines as well as correlations among cytokines and 99mTc-DTPA uptake. We found that only the three-injection dose of LPS induced significant BBB disruption in all sexes and strains. The treatment, strain, and sex, as well as treatment-by- strain and treatment-by-sex interactions significantly contributed to the variance. The mean brain/serum ratios of 99mTc-DTPA in the three-injection LPS group were ranked CD-1 male < CD-1 female < C57 male < C57 female. There were significant sex and strain differences in cytokine profiles in brain and blood, and pro-inflammatory cytokines and chemokines in brain were most strongly correlated with 99mTc-DTPA brain/serum ratios.

Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.

Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice.

Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc2155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc2155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination.

NK Cell Proportion and Number Are Influenced by Genetic Loci on Chromosomes 8, 9, and 17.

NK cells play a crucial role in innate immunity due to their direct cytotoxicity toward tumors, virally infected cells, and stressed cells, and they also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. In secondary lymphoid organs, NK cells compose the third most abundant lymphocyte subset after T cells and B cells. In this study, we perform an unbiased linkage analysis to determine the genetic loci that may limit the size of the NK cell compartment. Specifically, we exploit differences in NK cell proportion and absolute number between the C57BL/6 and the NOD mice. In addition to the previously identified linkage to chromosome 8, we find that a locus on chromosome 17, which encompasses the MHC locus, impacts NK cell number. Moreover, we identify a locus on mouse chromosome 9 that is strongly linked to the proportion and absolute number of NK cells. Using NOD congenic mice, we validate that both the MHC and the chromosome 9 loci influence the proportion and absolute number of NK cells. We have thus identified additional loci specifically linked to the proportion of NK cells and present some of the potential candidate genes comprised within these loci.

Mouse models of aerosol-acquired tularemia caused by Francisella tularensis types A and B.

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.

Rapid, quantitative mapping of anti-HIV type 1 envelope serum antibody specificities.

A new generation of extremely broad and potent neutralizing antibodies (bNAbs) has been isolated from HIV-infected subjects. This has refocused interest in the sites of vulnerability targeted by these bNAbs and in the potential for designing Envelope (Env) immunogens that display these sites. Standard methods for evaluating HIV-1 vaccine candidates do not enable epitope mapping on the HIV Env spike, the target for NAbs. To meet the need for rapid analysis of Ab specificity, we designed a multiplexed, quantitative mapping assay that can test for serum Ab competition for the binding of an HIV-1 Env gp120 to a panel of bNAbs directed to different sites of vulnerability on the Env that do not compete for one another in the assay. Using serum samples from rabbits immunized with various DNA prime/gp120 protein boost vaccines we were able to detect serum Ab competition for multiple classes of bNAbs in the postimmune samples that were significantly higher than background competition detected in samples obtained prior to vaccination. Importantly, application of this novel assay to our ongoing HIV-1 Env viral vector studies in mice has allowed us to distinguish qualitative differences in the Ab elicited by various regimens that ELISA cannot. Furthermore, pooled immunoglobulin from HIV-infected donors (HIVIg) competes for binding to the bNAb panel whereas a control pool from HIV-negative donors does not, highlighting the utility of this assay for human studies. This novel assay will add value in rational immunogen design and in the detailed, qualitative evaluation of binding and, potentially, neutralizing Abs elicited by natural infections and HIV-1 vaccine candidates.

[Morphofunctional changes of BALB/c and C57BL/6 mice's immune system under chronic bacterial gram-negative endotoxicosis].

Chronic endotoxicosis was modeled by subcutaneous injection of the sepharose in complex with LPS. In these conditions we have studied morphofunctional changes of the immune system of BALB/c and C57Bl/6 mice, which are characterized by the different types of the immune response (Th2 type is predominant in BALB/c, Th1--in C57Bl/6). In the 1st-7th day t in the serum of BALB/c mice the endotoxin level increased in 21.3 times, in C57Bl/6--in 20.6 times. The endotoxin antibodies significantly decreased in 1th-7th days, on the 14th day it increased in the serum of both mice's strains. Morphofunctional changes of the immune system after chronic endotoxicosis were different in BALB/c and C57BI/6 mice. On the 1th day after injection of LPS and sepharose, in the thymus of C57Bl/6 mice the cortex layer was exhausted because of cell death, in the thymus of BALB/c mice II-III stages of accidental involution were developed. On the 7-14th day after injection of LPS and sepharose in the spleen of C57Bl/6 mice T- and B-zones were hyperplastic, however in spleen of BALB/c mice only T-zone were enlarged. After LPS and sepharose injection changes of cytokine production synthesized by KonA activated splenic cells were found out. In both strains the level of proinflammatory cytokines--TNFalpha and IL-1beta decreased, as well the Th1-cytokine IL-2. The production o fTh2-cytokine - IL-4, significantly decreased only in C57BI/6 mice. We suggest that damaging effect of LPS injection is determined by predominant Th2 or Th2 types of the immune response.

Immunoadjuvant effects of hemagglutinating virus of Japan envelope (HVJ-E) on the inactivated H9 subtype avian influenza virus vaccine.

Avian influenza viruses (AIV) of the H9 subtype cause serious health problems in chickens, resulting in great economic losses to the poultry industry worldwide. The killed vaccine (KV) against H9 subtype AIV has been widely used in China since 1998 but has been linked with side effects in chickens and only partial protection. A few studies have demonstrated the immunostimulatory effects of the hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy. In this study, the adjuvant efficacy and the protective effects of HVJ-E, in combination with H9N2 AI KV against AIV were evaluated. The maturation of murine dendritic cells treated by HVJ-E was verified by FACS in the current experiment, then the antibody hemagglutination inhibition (HI) titers and cytokines and the post-challenge virological profiles (oropharyngeal and cloacal virus shedding) were investigated to define the immune responses in chickens. Our findings indicate that HVJ-E could induce dendritic cell (DC) maturation in mice. Injection of HVJ-E in chickens resulted in raised levels of IFN-ß and IFN-γ being present in sera suggesting a stimulatory effect in these animals. The antibody responses to AIV of chickens inoculated with HVJ-E adjuvanted killed H9-AIV were higher than those of chickens inoculated with oil adjuvanted H9-HIV. Furthermore, although inoculation of either HVJ-E or oil adjuvanted AIV reduced virus shedding following challenge, compared to controls, HVJ-E adjuvanted AIV was more effective in reducing shedding than oil adjuvant.

Anopheles stephensi saliva enhances progression of cerebral malaria in a murine model.

Malaria accounts for the greatest morbidity and mortality of any arthropod-borne disease globally. Recently, it was determined that the protective antisporozoite CD8+ T-cell response originates predominantly from cutaneous lymph nodes draining the site of parasite inoculation by an Anopheles mosquito. The female mosquito inoculates sporozoites along with an assortment of salivary proteins into the skin of its mammalian host. Mosquito saliva has demonstrable antihemostatic as well as various immunomodulatory activities, and studies with mosquito-borne viruses support a role for mosquito saliva in enhancement of transmission and exacerbation of disease. Early differences in immune response can be detected, which discriminate between mice that are resistant and susceptible to neurological pathology. This supports the idea that early divergence in the immune response may influence the likelihood of progression to the more severe forms of malaria. To evaluate the effect of mosquito feeding on the pathogenesis and immune response to malaria, we injected washed Plasmodium berghei sporozoites intradermally in the presence or absence of mosquito feeding. We observed that mice exposed to mosquito feeding in tandem with the inoculation of sporozoites had higher parasitemias and an elevated progression to cerebral malaria. This was associated with, in particular, elevated levels of interleukin-4 and interleukin-10, suppression of overall transcription in response to infection, and decreased extravasation of dendritic cells and monocytes. This study enhances to our understanding of the complexity of the interactions between the malaria parasite, its host, and the mosquito vector.

Measures of immune function of wild mice, Mus musculus.

The immune function of wild animals has been rather little studied. Wild animals' immune function may differ from that of laboratory bred animals because of their different environments. This idea follows from the concept of resource partitioning in which animals distribute scarce resources to all aspects of life, including to costly immune responses. A logical extension of this idea is that there may be substantial interindividual variation in the immune function of wild animals. To begin to investigate this, we compared the immune function of a laboratory bred mouse strain (C57BL/6, a widely used mouse strain that makes potent immune responses) and wild caught Mus musculus. We found that by most measures of immune function, the wild caught mice had greater immune function. Specifically, wild mice had greater concentrations and more avid antigen-specific IgG responses, as well as higher concentrations of total IgG and IgE, compared with those laboratory bred mice. Moreover, flow cytometric analysis showed a comparatively greater overall level of activation of the cells of the immune system in wild mice. Lastly, we observed that immune function was substantially more variable among wild caught mice than among the laboratory bred mice. The next research challenge is to understand which aspects of an individual animal's life determine its immune function.

Morphofunctional characteristic of the immune system in BALB/c and C57BL/6 mice.

Inbred animals serve as an adequate model to study the role of genetic factors in adaptive, disadaptive, and pathological processes. Morphofunctional study of the immune system was performed on intact BALB/c and C57Bl/6 mice. The structural and functional parameters of the immune system in BALB/c and C57Bl/6 mice differ under physiological conditions. In BALB/c mice, volume density of T zone in the spleen and production of IL-2, IL-3, IL-4, IL-10, and TNF-α were much higher than in C57Bl/6 mice. However, IL-12 production in BALB/c mice was lower than in C57Bl/6 mice. C57Bl/6 mice were characterized by higher cytostatic activity of splenic NK cells. The observed interstrain differences are genetically determined and contribute to the type of adaptive processes and different sensitivity of these mice to pathogenic agents.

Morphofunctional characteristics of the immune system in CBA and C57Bl/6g mice.

We studied peculiarities of morphofunctional organization of the immune system in C57Bl/6g and CBA mice differing by their susceptibility to various types of infectious agents. The revealed differences in the structure of lymphoid organs, T lymphocyte subpopulation ratio and their differentiation into Th1/Th2 cells after mitogen stimulation drove us to a conclusion on genetically determined regularities in the development of the immune response in these animal strains.

Interleukin-17-mediated control of parasitemia in experimental Trypanosoma congolense infection in mice.

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.

Family relationship of female breeders reduce the systematic inter-individual variation in the gut microbiota of inbred laboratory mice.

The gut microbiota (GM) may influence disease expression in several animal models for inflammatory diseases. It may therefore seem reasonable to pursue reduction in the number of animals used for individual studies by reducing the variation in the GM. Previous studies have shown that the composition of the GM is related to genetics to a certain extent. We hypothesized that the GM similarity in a group of mice born by mothers not being sisters would be lower than that in a group born by mothers being sisters. The lower similarity could lead to clustering of the GM of mice born by non-sisters according to their mothers, while such clustering would not be visible if the mothers were sisters. We used 16S rRNA gene (V3 region) polymerase chain reaction-derived amplicon profiling by denaturing gradient gel electrophoresis (DGGE) to study the GM composition in caecum samples of 33 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were sisters, as well as caecum samples of 35 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were not sisters. Principal component analysis revealed a significant difference between the litters from the breeding set-up with dam breeders that were not sisters, whereas no significant difference between the litters based on the breeding set-up with dam breeders that were sisters was observed. The results obtained indicate that the systematic variation in the GM of inbred mice can be reduced by increasing the family relatedness of the breeding pairs.