Sex differences matter in the gut: effect on mucosal immune activation and inflammation
© Sankaran-Walters et al.; licensee BioMed Central Ltd. 2013
Received: 15 January 2013
Accepted: 11 April 2013
Published: 7 May 2013
Women and men have diverse responses to many infectious diseases. These differences are amplified following menopause. However, despite extensive information regarding the effects of sex hormones on immune cells, our knowledge is limited regarding the effects of sex and gender on the function of the mucosal immune system. Sex differences also manifest in the prevalence of gut associated inflammatory and autoimmune disorders, including Crohn’s disease, ulcerative colitis and Celiac disease. It is thus hypothesized that a baseline sex-associated difference in immune activation may predispose women to inflammation-associated disease.
Peripheral blood samples and small intestinal biopsies were obtained from 34 healthy men and women. Immunophenotypic analysis of isolated lymphocytes was performed by flow cytometry. Oligonucleotide analysis was used to study the transcriptional profile in the gut mucosal microenvironment while real-time PCR analysis was utilized to identify differential gene expression in isolated CD4+ T cells. Transcriptional analysis was confirmed by protein expression levels for genes of interest using fluorescent immunohistochemistry. Data was analyzed using the GraphPad software package.
Women had higher levels of immune activation and inflammation-associated gene expression in gut mucosal samples. CD4+ and CD8+ T cells had a significantly higher level of immune activation-associated phenotype in peripheral blood as well as in gut associated lymphoid tissue along with higher levels of proliferating T cells. CD4+ T cells that showed upregulation of IL1β as well as the TH17 pathway-associated genes contributed a large part of the inflammatory profile.
In this study, we demonstrated an upregulation in gene expression related to immune function in the gut microenvironment of women compared to men, in the absence of disease or pathology. Upon closer investigation, CD4+ T cell activation levels were higher in the LPLs in women than in men. Sex differences in the mucosal immune system may predispose women to inflammation-associated diseases that are exacerbated following menopause. Our study highlights the need for more detailed analysis of the effects of sex differences in immune responses at mucosal effector sites.
Sex and gender-related differences are reflected in the varying magnitude of inflammatory or autoimmune diseases in men and women and are attributed to the diversity of immune responses to infectious or non-infectious stimuli in men and women . Women have a significantly higher incidence of autoimmune diseases than men which are mediated by humoral and adaptive immune responses . It is likely that some of these effects in women are due to the immune-modulatory effects of sex hormones [2–4].
Sex hormones play an important role in the development of adaptive cellular immunity, especially following repeated antigenic stimulus . Gene expression profiling of immune cells from women detected an increased expression of genes associated with inflammatory and cytotoxic T cell responses . However, other studies reported contrasting findings showing increased expression of immune response-related genes in men as compared to women . Discordance among these findings might have been influenced by the timing of the sample collection and the hormonal milieu in women. Sex-specific immune modulation is likely mediated by sex hormones through activation of their specific receptors. Estrogen receptors are expressed on T cells, macrophages and other immune cells that are localized in several tissue compartments in addition to the reproductive tract [7–10]. Estrogen directly modulates immune function through cytokine production, cell activation, and cell proliferation [7–10]. Estrogen is also shown to influence the immune response to tissue injury [11, 12]. While an enhanced immune response can be protective against some infections, this could also predispose women to more autoimmune disease . Sex-related differences in antibody production, lymphocyte proliferation, and cytotoxic activity and the influence of steroid hormones on these functions has been previously investigated . Peptide hormones including prolactin, luteinizing hormone (LH), and follicle stimulating hormone (FSH) enhance proliferation of lymphocytes in response to mitogenic stimuli.
Estrogen and progesterone stimulate their cytotoxic activity by enhancing the release of reactive oxygen species [14, 15]. Recent gene expression analysis of isolated peripheral blood mononuclear cells (PBMC) from men and women showed a markedly increased expression of immune response related genes in women. Levels of inflammatory and cytotoxic response-associated genes, including IFNγ, IL12Rβ2 and (Leukotriene) LTβ, were all increased in women compared to men . These findings suggest sex-specific differences in T cell function and regulation.
Despite extensive information regarding the effects of sex hormones on immune cells, our knowledge is limited regarding the effects of sex and gender on the function of the gut mucosal immune system [14, 15]. Most investigations focused on the analysis of the peripheral blood compartment. However, the gut-associated lymphoid tissue (GALT) is the largest lymphoid organ in the body and is an important site for host-microbe interactions. The involvement of the GI tract is well documented in several inflammatory and autoimmune disorders, including Crohn’s disease, ulcerative colitis, and Celiac disease. It is not known whether the regulation of intestinal inflammatory response is influenced by sex differences . Differences in immune cell infiltration of the colonic mucosa such as mast cells have been shown to correlate with symptomatic differences between the sexes in Irritable Bowel Syndrome (IBS) . However, no differences were observed in healthy controls . The reason for observed gender differences has been attributed to sex hormones and their effects on mast cells, but little is known regarding the effects of sex on lymphocyte function . While many of these studies have been conducted in older women in whom sex hormone levels are low, little is known about sex differences between women and men of younger age groups and if these sex differences play a role in the development of inflammation-associated diseases later on in life.
While studies in the colon have provided evidence for the role of sex in mucosal immune responses, the jejunum is one of the most cellular regions of the small intestine with a large percentage of the body’s T cells. CD4+ T cells are the cellular targets of viral infections such as HIV where gender differences and sex hormones play an important role in pathogenesis and transmission kinetics. Thus, the goal of this study was to investigate the link between sex differences and the regulation of intestinal inflammation by analyzing jejunal biopsies and peripheral blood samples from healthy age-matched male and female participants. Since the gut mucosa is a site of interaction between the gut microbiome and the host immune system, we investigated the effect of sex differences on transcriptional profiles of gut biopsies as well as isolated mucosal CD4+ T cells. Our findings suggest that the sex differences manifest as higher levels of immune activation and inflammation in women compared to men in the absence of any disease or pathology, thus influencing gut mucosal immune response and function.
Healthy female participants were enrolled in the study at the Center for AIDS Research, Education, and Services clinic and at the Kaiser Permanente Clinic in Sacramento, CA. Male participants were enrolled from the same general geographical area as the female participants. The inclusion criteria included: age between 20 and 60, healthy, and able to participate in an upper GI endoscopy procedure with minimal risk. The exclusion criteria for all participants included: incidence of cardiovascular disease, diabetes, smoking, excessive alcohol consumption, use of illicit drugs or medications. Exclusion criteria specific to female participants was a peri- or post-menopausal state as defined by SWAN . Samples were obtained during the follicular phase of the menstrual cycle (4–7 days following last menstrual period). Menopause is associated with changes in mucosal epithelial function which can further augment observed gender based differences in immune function. Thus postmenopausal state was an exclusion criterion. Age also plays a role in immune responses and to minimize the effects of age all participants enrolled were under the age of 55. Over 40 potential participants were reviewed by the research team and 34 fulfilled the enrollment criteria. The study protocol was approved by the UC Davis Institutional Review Board. All participants provided written, signed informed consent. Twenty milliliters (mL) of blood was collected by venipuncture in EDTA-containing tubes and processed at each time point. Pinch biopsies were obtained by upper GI endoscopy from the jejunum under conscious anesthesia according to previously published protocols . All samples were collected between 7:00 AM and 9:00 AM at the UCD Gastroenterology Clinic, Sacramento, CA after overnight fasting.
Isolation of peripheral blood mononuclear cells (PBMC)
Whole blood was collected in EDTA-containing tubes and centrifuged for 20 minutes. The white blood cell buffy coat was collected, diluted in 1X PBS, layered over ficoll gradients (Atlanta Biological, Lawrenceville, GA), and centrifuged 20 minutes. The mononuclear cell layer was collected and pelleted by centrifugation. Cells were cultured overnight at 37°C, 5% C02 in culture media (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, and 1% L-glutamine (Gibco) . Five to seven million PBMC were used for CD4+T cell isolation.
Isolation of intestinal lymphocytes
Jejunum biopsies were placed in isolation media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin (Gibco, Grand Island, NY) and collagenase type IIb (1 mg/mL) (Sigma, St. Louis, MO)  and subjected to shaking at 37°C for four periods of 30 minutes. Cells were washed with RPMI media and rested overnight in culture media (RPMI 1640 supplemented with 15% FBS, 1% gentamycin, 100 U/mL penicillin, 100 U/mL streptomycin, 1% L-glutamine, 1% sodium pyruvate, 2.5% Hepes, and 0.5% amphotericin B (Gibco)) at 37°C with 5% CO2. Cells were filtered through a 40 μm filter the next day and counted. Three to four million lymphocytes were filtered the same day for further CD4+T cell isolation.
CD4+ T cell isolation
Isolated lymphocytes from the jejunum and peripheral blood were enriched for CD4+T cells by magnetic bead sort (Dynal, Carlsbad, CA). Magnetic beads pre-coated with CD8 antibody were incubated with isolated lymphocytes for 30 minutes at 4°C on a gentle rotating platform and CD8 lymphocytes were collected by magnet and incubated with CD13 coated magnetic beads. Pan-mouse IgG beads (Dynal) were first incubated for 30 minutes at 4°C on a rotating platform with monoclonal IgG CD13 antibody (BD, San Jose, CA), and then CD13 coated beads were incubated with CD8 depleted lymphocytes for 30 minutes at 4°C on a gentle rotating platform. The CD8- CD13- lymphocytes were collected by magnet, washed in culture media, and incubated overnight at 37°C/5%CO2 at a maximum concentration of 2 million cells/ml. Cells were pelleted the following day and stored in -80°C until use. Supernatant from overnight culture was also stored at -80°C until needed. Purity of PBMC (90-100%) and jejunal lymphocytes (65-85%) was determined by flow cytometry.
Isolated lymphocytes (1 million cells/tube) were incubated with Aqua (Amcyan) Live/Dead Dye (Invitrogen, Carlsbad, CA) (0.5 uL/mL 1XPBS + 1million cells). Lymphocytes were then washed with staining buffer (filter sterilized 1XPBS with 3% heat-inactivated FBS and 0.1% sodium azide). Cells were incubated with 10% normal mouse serum (Jackson Immunoresearch Laboratories, Inc) and then with fluorescent mouse anti-human monoclonal antibodies. CD3 (Beckton Dickinson, Mountain View, CA), CD45RA, CD45RO, CD8 (Invitrogen), CD95 (Biolegend, San Diego, CA), CD28, CD4, HLADR, Ki67 (E-Bioscience) antibodies were used. Cells were washed and fixed with 1% paraformaldehyde (PFA) (Sigma) prior to analysis on the LSR II flow cytometer (Beckton Dickinson at the UC Davis core facility). The cells were first gated based on negative staining with live dye, followed by a lymphocyte gate. These populations were further analyzed by specific staining. A minimum of 500,000 events was collected per sample and data was analyzed using Flow Jo (Tree Star, Inc. San Carlos, CA).
Isolated lymphocytes (~1 million cells) were stimulated with phorbol 12-myristate 13-acetate (PMA) (1 ng/mL) + Ionomycin (1 μM/mL) (Sigma), or negative control media alone at 37°C with 5% CO2 for 6 hours. Anti-CD28 pure monoclonal antibody (Ebioscience) was added to aid co-stimulation, followed by addition of Brefeldin A (Sigma) for the final 5 hours to prevent Golgi transport of cytokines out of cells. Cells were washed with 1X PBS/1%BSA, stained for surface markers, and fixed in 1% PFA. Fixative was washed out and cells were permeabilized with Caltag Permeabilization Solution B (Invitrogen) and stained with monoclonal antibodies IFNγ, IL-2, and TNF-α (BD, Ebioscience) for 20 minutes at room temperature. Samples were washed and fixed with 1% PFA before analysis with LSR II. A minimum of 500,000 events were collected for each sample and analyzed using Flow Jo software. Polyfunctional cell analysis was performed using SPICE (v4.1.6) and PESTLE (v1.5.4) software courtesy of Mario Roederer, Vaccine Research Center, NIAID/NIH, Bethesda, MD.
Gene expression profiles in gut biopsies from participants were analyzed using microarray technology and real-time PCR. In brief, total RNA was isolated from cryopreserved jejunal tissue utilizing protocols and reagents in the RNeasy RNA isolation kit (Qiagen). Messenger RNA amplification, labeling, hybridization to human whole-genome U95av2 GeneChips© (Affymetrix) staining and scanning were performed as previously described [21, 22] utilizing kits and protocols described in the Affymetrix Gene Expression Analysis Technical Manual. Microarray data were analyzed cross-sectionally amongst all groups utilizing the dChip (http://www.hsph.harvard.edu/cli/complab/dchip/) software program. A minimum fold-change of 1.5 (p-value ≤ 0.05) between groups was established as a cut-off criterion for statistical validation. Samples from male participants were used as baseline values for identifying differentially expressed genes (DEG) in samples from female participants. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) software.
RNA was also isolated from CD4+ T cells enriched from PBMC as well as LPL using the same methods as in the microarray analysis. CDNA was synthesized using Superscript III (Invitrogen, CA) and used in Taqman© Real-Time PCR assay as previously described (Applied Biosystems, CA) [21, 22]. The data were normalized to the housekeeping gene GAPDH and calibrated to the average of the male samples from the same tissue source. The data were presented as a fold increase in RNA level compared to the calibrator .
Immunohistochemical analysis (IHC) was performed to detect and localize expression of IL17, TNFα, FoxP3 and IL1β in the small intestinal mucosa. Jejunal biopsies were embedded in paraffin blocks following fixation with 4% PFA. Tissue sections (5 μm) were incubated with polyclonal anti-IL17, anti-TNFα, anti-FoxP3 or anti-IL1β overnight at 4°C, followed by incubation with FITC-labeled secondary antibodies for 1 hour as previously described. Sections were mounted using SlowFade with Dapi (Invitrogen, Carlsbad, CA). Images were captured by confocal laser microscopy using LSM 5 and PASCAL software (Zeiss, New York) and then analyzed using Image J software, NIH, Maryland.
Data sets were analyzed using the two-tailed paired or unpaired t-test as applicable, taking into account the distribution of the data (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, http://www.graphpad.com). Due to the limited availability of samples to perform all the analyses, a selected number of samples were utilized in each assay over 75% overlap between most experiments. The real-time data were analyzed for statistically significant differences at the level of the calibrated values as well as at the level of the linearized fold change representing relative expression.
Demographics of study participants
Characteristics of participants
Age Range (yrs)
Baseline gut mucosal gene expression shows increased immune activation and inflammation in women compared to men
Women have higher levels of T cell proliferation and T cell activation in peripheral blood and gut of women in the absence of disease
The percentage of T cells expressing the activation marker, HLA-DR, was determined using flow cytometry (Figure 5B). The percentage of activated CD4+ T cells in peripheral blood of women was significantly higher than in men (p=.0014). The percentage of HLA-DR expressing activated CD8+ T cells was also increased in women compared to men with a mean difference of 19.23 (p=.0006). This increase in T cell activation in peripheral blood is also reflected in GALT. The percentage of cells that were CD4+ HLA-DR+ in the LPL was also higher in women compared to men, indicating that cell activation in women was higher than in men (p=.0195), and CD8+ T cell activation was also higher in women compared to men (p=.0078). A positive correlation (p>.05) was observed between CD4+ HLADR and CD8+ HLADR expression in all groups in PBMC as well as LPL.
LPL derived CD4+ T cells from women are also more sensitive to non-specific stimulation with PMS/Ionomycin as compared to men. LPL and PBMC were stimulated with PMA/Ionomycin and TNFα levels were measured by intracellular staining and flow cytometry (Figure 5C). In general, a higher level of responsiveness was observed in LPLs of all study participants, male and female. However, CD4+ cells from women had higher levels of TNFα in the LPLs than in men (41.61% vs 28.8%) (p<0.05). No significant differences were observed in CD8+ T cells from the blood or gut. Surprisingly, the percentages of CD8+ T cell producing TNFα on stimulation in the gut was significantly lower than that in the peripheral blood in women, but not in men. Increased expression of TNFα protein was also detected the gut tissue of women by immunohistochemistry (Figure 3A, B and C).
Gut mucosal CD4+ T cells contribute to the elevated Th17 associated inflammatory profile in women
The CD4+ T cells also showed a significant increase in expression of FOXP3 as well as TGFβ in women compared to men (p<.05) (Figure 7). In both cases the LPLs tended to have higher expression than PBMC-derived CD4+ T cells, but this increase was not statistically significant. These observations could be due to a compensatory response to increased immune activation in CD4+ cells. IHC for Fox P3 demonstrated significantly higher numbers of FOXP3+ cells in villi of women compared to men as well as increased expression in positive cells (Figure 7C, D, E).
Sexual dimorphism of the immune system plays an important role in disease occurrence, host-microbe interactions, and mechanisms of pathogenesis . Females respond to infections with a more robust innate and adaptive immune response compared to males. This, however, is a double-edged sword. For some infections, this can be beneficial because it results in faster clearing of the pathogen. In others, such as H1N1 influenza virus, the robust immune response may result in a greater number of fatalities in females compared to males. Increased immune activation may potentially place females at a greater risk of developing autoimmune disorders.
In this study, we demonstrated an upregulation of gene expression related to immune function in the gut microenvironment of women compared to men in the absence of any current disease or pathology. Upon closer investigation, CD4+ T cell activation levels were higher in the LPLs in women than in men. Prior studies have demonstrated an increased immune response in women following nonspecific stimulation of the peripheral blood . However, the gut mucosal immune system is the largest in the body. The gut is also the most important site of host-microbe interactions. It is likely that the heightened responsiveness in the gut is triggered by the need to reduce the systemic load of microbial translocation and may be driven by gender associated differences in that process. Our study demonstrated sex differences in the mucosal immune system in which women had a higher baseline level of immune activation compared to their male counterparts thus predisposing them to inflammation-associated diseases that are exacerbated following menopause.
Studies in murine models of burn injury indicate that higher estrogen levels in female mice contribute to improper cell-mediated immune response to injury with an aberrant inflammatory response that increases mortality. In contrast, male mice are immunosuppressed following hemorrhagic injury and are more likely to suffer from sepsis than female mice with a comparatively enhanced cellular immune response under the same conditions . In our study, healthy women had an enhanced inflammatory response (IL1β pathway) as well as an immune proliferative response (IL12 pathway) in the small intestinal mucosa, which may be protective in some cases of injury. Furthermore, an increase in TNFα and IFNγ, likely to originate from the T cells in the LPLs following mitogenic stimulation, was evidence of a robust cellular immune response in women as compared to men in our samples. This occurred both at the level of the gut microenvironment as well as the cellular level in CD4+ T cells.
Prior studies reported that following mitogenic stimulation, IL17A expression was upregulated in men compared to women in PBMC . In our study, we showed a steady state increase of IL17A expression in CD4+ T cells of women compared to men. IL17A is produced by both CD4+ and CD8+ T cells. In a limited number of patient samples, increased IL17 MFI following mitogenic (PMA/Ionomycin) stimulations was detected in PBMC of men than of women (data not shown). In CD4+ T cells, expression was increased of transcription factors upstream of IL17A acivation, such as RORC and STAT3, as well as downstream effectors such as iNOS and Lipocalin. Th17 responses in infectious diseases, such as HIV infection, are important in controlling mucosal pathogenesis. In animal models, basal Th17 levels may play a role in disease progression. A basal gender-based difference in TH17 expression may contribute to gender differences observed in disease progression in viral and bacterial infection. This increase in baseline cellular immune response could be protective during infections but the lack of resolution may be detrimental in the development of autoimmune diseases.
Estradiol in women plays an important role in the repair and regeneration of mucosal surfaces. In healthy women, the Wnt signaling pathway is upregulated, indicating an increase in turnover and regeneration of the gut mucosa, likely of the epithelial cells. Wnt/β Catenin signaling is important for the differentiation and maturation of gut epithelial stem cells into mature enterocytes as well as Paneth cells in crypts. The increase in expression of transcripts belonging to this pathway could also support the concept of increased proliferation and turnover of epithelial cells in women as compared to men. This may also indicate baseline differences in epithelial integrity between males and females. These findings warrant further investigation.
Despite the small sample size in our study, it has identified inherent differences in the gut mucosal immune system based on the sex and gender of the participant and has pointed to an important concept relevant to large-scale patient studies of drug efficacy or immune modulators. It was not feasible to perform all cellular and molecular analyses on all samples due to limitations associated with sample collection. It was beyond the scope of this study to identify latent viral infections such as EBV and CMV that may drive immune activation in health adults and can be investigated in future studies. The gut microbiome also plays an important role in the immune homeostasis. While microbiome differences may exist between the sexes, such studies have not been performed in detail. Thus, similar studies using animal models may be necessary to further delineate the effects of sex on gut mucosal function. Our study is the first study to demonstrate the baseline differences in the function of GALT, the largest lymphoid tissue in the body.
Taken together with the knowledge that sex-based differences in the immune system play an important role in disease progression in infectious disease and vaccine responses, sex of the study population should be considered during clinical trials . Our study highlights the need for more extensive and detailed analysis of the effects of sex differences in immune responses at mucosal effector sites.
We acknowledge the UC, Davis SOM Microarray Core Facility for technical support. This study was supported by grants from the National Institute of Health (NIH) R01 (DK61297, AI43274). Dr. Sankaran is supported by a Building Interdisciplinary Research Careers in Women’s Health award (K12 HD051958) funded by the NICHD, ORWH, and the NIA.
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