The Female Lower Genital Tract Is a Privileged Compartment with IL-10 Producing Dendritic Cells and Poor Th1 Immunity following Chlamydia trachomatis Infection

While a primary genital tract infection with C. trachomatis stimulates partial-protection against re-infection, it may also result in severe inflammation and tissue destruction. Here we have dissected whether functional compartments exist in the genital tract that restrict Th1-mediated protective immunity. Apart from the Th1-subset, little is known about the role of other CD4+ T cell subsets in response to a genital tract chlamydial infection. Therefore, we investigated CD4+ T cell subset differentiation in the genital tract using RT-PCR for expression of critical transcription factors and cytokines in the upper (UGT) and lower genital tract (LGT) of female C57BL/6 mice in response to C. trachomatis serovar D infection. We found that the Th1 subset dominated the UGT, as IFN-γ and T-bet mRNA expression were high, while GATA-3 was low following genital infection with C. trachomatis serovar D. By contrast, IL-10 and GATA-3 mRNA dominated the LGT, suggesting the presence of Th2 cells. These functional compartments also attracted regulatory T cells (Tregs) differently as increased FoxP3 mRNA expression was seen primarily in the UGT. Although IL-17A mRNA was somewhat up-regulated in the LGT, no significant change in RORγ-t mRNA expression was observed, suggesting no involvement of Th17 cells. The dichotomy between the LGT and UGT was maintained during infection by IL-10 because in IL-10-deficient mice the distinction between the two compartments was completely lost and a dramatic shift to the predominance of Th1 cells in the LGT occurred. Unexpectedly, the major source of IL-10 was CD11c+ CD11b+ DC, probably creating an anti-inflammatory privileged site in the LGT

Mucosal Vaccine Development Based on Liposome Technology

Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen. Hence, there is an increasing demand for vaccine formulations that can induce strong protective immunity following oral, respiratory, or genital tract administration. At present, only few mucosal vaccines are found on the market, but recent technological advancements and a better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimistic view on the future of mucosal vaccines. Compared to live attenuated vaccines, subcomponent vaccines, most often protein-based, are considered safer, more stable, and less complicated to manufacture, but they require the addition of nontoxic and clinically safe adjuvants to be effective. In addition, another limiting factor is the large antigen dose that usually is required for mucosal vaccines. Therefore, the combination of mucosal adjuvants with the recent progress in nanoparticle technology provides an attractive solution to these problems. In particular, the liposome technology is ideal for combining protein antigen and adjuvant into an effective mucosal vaccine. Here, we describe and discuss recent progress in nanoparticle formulations using various types of liposomes that convey strong promise for the successful development of the next generation of mucosal vaccines

Lower genital tract DC are the primary source of IL-10 production following <i>C. trachomatis</i> infection.

<p>(A) Lymphocytes from the lower genital tracts (LGT) from naïve WT mice or <i>C. trachomatis</i> infected (day 24) were isolated and sorted into CD4, CD8α, MΦ, pDC, and cDC populations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001179#s4" target="_blank">Materials and Methods</a> for definitions). (B) The relative level of IL-10 mRNA expression in the indicated sorted populations was analyzed by RT-PCR on day 24 of infection. Values were normalized against the housekeeping gene (HPRT) and given as mean normalized expression ± SEM, from a representative of 2 experiments, each with 3 samples per group and 3 pooled mice in each sample. (C, left panel) Immunohistochemistry was used to confirm the presence of IL-10 protein (TxRed; red), CD11c (FITC;green) and Topro-3 (blue) on day 24 of infection. (C, right panel) IL-10 levels from the LGT of naïve mice or after 24 days of infection were measured using an ELISA. (D) The numbers of sorted cDC per mouse from the LGT of naïve mice, and on day 10 and 24 of infection (left panel). IL10 mRNA expression normalized to HPRT in the sorted cDC from the LGT (middle panel) and UGT (right panel) of naïve mice, and on day 10 and 24 of infection. (E) IL10^−/− or WT CD4⁺ T cells were adoptively transferred to nu/nu mice and IL10 mRNA expression in the LGT (left panel) or upper genital tract (UGT; right panel) was assessed in unchallenged mice or after 24 days of infection.</p

T-bet dominates the upper genital tract, while GATA-3 is upregulated in the lower genital tract.

<p>Regional lymph nodes (PALN and ILN), lower (LGT) and upper (UGT) genital tracts were collected from naïve and <i>C. trachomatis</i> infected mice at the indicated time points. RT-PCR was undertaken to determine the mRNA levels of (A) T-bet, (B) GATA-3 and (C) RORγ-t expression, normalized to the housekeeping gene (A-D) HPRT or CD3-γ (E). (A-C,E) Data is expressed as normalized mean expression ± SEM, or (D) fold-increase over naïve levels. Values are from one representative experiment of 3 giving similar results, and 5 mice per time point. * p<0.05, ** p<0.01.</p

The dichotomy between upper and lower genital tract remains regardless of infectious dose.

<p>(A) The chlamydial inclusion forming unit (IFU) load in the upper (UGT) versus lower (LGT) genital tract was determined by culture of tissue homogenates on day 5, 15 and 24 of infection. Results are expressed as mean log₁₀IFU/ml ± SEM from 4 mice per time point. (B) Mice were infected with 10⁶, 10⁵<i>C. trachomatis</i> EBs or mock-infected with 10⁶ heat-killed (HK EBs). IFN-γ mRNA expression levels in the LGT and UGT on day 15 and 24 of infection were determined using RT-PCR. Results are expressed as mean expression normalized to the housekeeping gene, HPRT, ± SEM from 4 mice per time point.</p

Distinct cytokine mRNA profiles in different regions of the genital tract during <i>C. trachomatis</i> infection.

<p>(A) sections of upper genital tract (UGT; left panels) and lower genital tract tissue (LGT; right panels) were stained for CD4⁺ T cells on day 3, day 5, and day 10 post-infection, as indicated. Representative sections from one experiment of 3 giving similar results are shown. Large photographs are 10× magnification with 20× inset. (B) Cytokine mRNA levels were determined using RT-PCR, in the LGT and UGT at the indicated time points of infection. Results are expression of IFN-γ, IL-17A, IL-10 and IL-4 mRNA normalized to the housekeeping gene, HPRT, ± SEM. Data is from a representative experiment out of 3 giving similar results, and 5 mice per time point. (C) Tissue sections from the UGT (upper panel) and LGT (lower panel) were stained for CD4 (TxRd; red), IFN-γ FITC; green) and Topro-3 (blue) on day 24 of infection. Cytokine bead array analysis was used to quantify the production of IFN- γ following PMA/ionomycin <i>in vitro</i> stimulation of CD4⁺ sorted T cells from infected mice on day 24 (right panel). (D) PMA/ionomycin stimulated CD4⁺ T cells from the LGT were stained for IL-4 (upper panel) or IL-5 (lower panel) and analysed by flow cytometry.* p<0.05, ** p<0.01.</p