Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

Induction of Protective Immunity against Chlamydia muridarum Intravaginal Infection with a Chlamydial Glycogen Phosphorylase

We evaluated 7 C. muridarum ORFs for their ability to induce protection against chlamydial infection in a mouse intravaginal infection model. These antigens, although encoded in C. muridarum genome, are transcriptionally regulated by a cryptic plasmid that is known to contribute to C. muridarum pathogenesis. Of the 7 plasmid-regulated ORFs, the chlamydial glycogen phosphorylase or GlgP, when delivered into mice intramuscularly, induced the most pronounced protective immunity against C. muridarum intravaginal infection. The GlgP-immunized mice displayed a significant reduction in vaginal shedding of live organisms on day 14 after infection. The protection correlated well with a robust C. muridarum-specific antibody and a Th1-dominant T cell responses, which significantly reduced the severity but not overall incidence of hydrosalpinx. The GlgP-induced partial protection against upper genital tract pathology suggests that GlgP may be considered a component for a multi-subunit vaccine. These results have demonstrated that intramuscular immunization of mice with purified proteins can be used to identify vaccine antigens for preventing intravaginal infection with C. trachomatis in humans

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1x10(2), 1x10(4), and 1x10(6) inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4(+) and CD8(+) cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4(+) and CD8(+) cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4(+) and CD8(+) cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1x10(4), and 1x10(6) IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections

Antibody signature of spontaneous clearance of Chlamydia trachomatis ocular infection and partial resistance against re-challenge in a nonhuman primate trachoma model.

Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. Here, we investigate whether protracted clearance of a primary infection in nonhuman primates is attributable to antigenic variation or related to the maturation of the anti-chlamydial humoral immune response specific to chlamydial antigens.Genomic sequencing of organisms isolated throughout the protracted primary infection revealed that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. To explore the maturation of the humoral immune response as a possible reason for delayed clearance, sera were analyzed by radioimmunoprecipitation using intrinsically radio-labeled antigens prepared under non-denaturing conditions. Antibody recognition was restricted to the antigenically variable major outer membrane protein (MOMP) and a few antigenically conserved antigens. Recognition of MOMP occurred early post-infection and correlated with reduction in infectious ocular burdens but not with infection eradication. In contrast, antibody recognition of conserved antigens, identified as PmpD, Hsp60, CPAF and Pgp3, appeared late and correlated with infection eradication. Partial immunity to re-challenge was associated with a discernible antibody recall response against all antigens. Antibody recognition of PmpD and CPAF was destroyed by heat treatment while MOMP and Pgp3 were partially affected, indicating that antibody specific to conformational epitopes on these proteins may be important to protective immunity.Our findings suggest that delayed clearance of chlamydial infection in NHP is not the result of antigenic variation but rather a consequence of the gradual maturation of the C. trachomatis antigen-specific humoral immune response. However, we cannot conclude that antibodies specific for these proteins play the primary role in host protective immunity as they could be surrogate markers of T cell immunity. Collectively, our results argue that an efficacious subunit trachoma vaccine might require a combination of these antigens delivered in their native conformation

Identification of trachoma conserved antigens by western blotting using chlamydial antigen specific antibodies.

<p>IP was performed using monkey RML134 serum and chlamydial proteins prepared from non-denaturing detergent lysed non-radiolabeled A2497 infected McCoy cells. The IP proteins we identified by Western blot using a panel of rabbit monospecific antibodies generated against PmpD and mouse monoclonal antibodies specific to CPAF and Pgp3. (A) The higher MW polypeptides were identified as full length PmpD (155 kDa) and its proteolytically processed 82 kDa translocator and 73 kDa passenger domains <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002248#pntd.0002248-Swanson1" target="_blank">[10]</a> (B) Two lower MW polypeptides reacted with antibodies specific to CPAF and Pgp3.</p

Antibody signature of <i>C. trachomatis</i> infected macaques following primary infection and re-challenge.

<p>Three monkeys were infected ocularly with <i>C. trachomatis</i> strain A2497 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002248#pntd.0002248-Kari2" target="_blank">[7]</a>. Monkeys cleared the infection 9–14 wpi (RML134: 13 wpi; RML124: 8 wpi; RML126: 14 wpi). Clinical disease remained severe to moderately severe during the periods of culture positivity and continued until about 30 wpi. Three months after resolution of disease monkeys were re-challenged and all monkeys exhibited partial protection characterized by a more rapid clearance of organism (3–7 wpi) with an accompanying reduction in the intensity and duration of ocular inflammation (11 wpi). Sera collected from monkeys during primary infection and following re-challenge were analyzed by RIP using intrinsically labeled chlamydial proteins prepared from non-denaturing detergent lysed infected McCoy cells. Sera from all three macaques produced a similar yet simple antigen recognition profile that differed in complexity over the period of primary infection and re-challenge. A total of 10 proteins were immunoprecipitated with molecular masses of 166, 90, 72, 60, 40, 29, 25, 23, 16 and 15 kDa. There was a noticeable temporal and recall recognition pattern of the antigens in all three monkeys. The MOMP (40 kDa) recognition was strong very early post infection (2–4 weeks) and consistently sustained throughout the entire period of infection and disease. In contrast immunoprecipitation of the other chlamydial proteins occurred later following primary infection, with the strongest immunoprecipitation reactions occurring at the time of infection eradication and disease resolution. There was a recognizable increase in the immunoprecipitation of all of these proteins by all three animals following re-challenge.</p

The majority of chlamydial antigens recognized during primary infection and re-challenge are shared between trachoma serovars.

<p>RIP was performed using lysates made from <i>C. trachomatis</i> serovars A2497 and Ba. Serum from monkey RML134 was used in the experiment and four representative time points throughout the course of infection and re-challenge were analyzed. Antibody to MOMP was serovar-specific, reacting most intensely with the infecting A2497strain. Conversely, antibody reactivity against the other immunogenic proteins was cross-reactive between trachoma serovars demonstrating that these antigens are conserved.</p

A majority of the antibodies generated against chlamydial specific and conserved antigens recognize conformational determinants.

<p>(A) RIP was performed with sera from all three infected NHP obtained from a single time point 13 weeks after re-challenge using A2497 infected cell lysates that were treated at 70°C for 30 min or kept at ambient temperature. Heat treatment of chlamydial antigens partially destroyed antibody recognition of MOMP in all three monkeys. Heating also destroyed or partially destroyed the antigenicity of the majority of the other IP antigens indicating that antibodies generated against the antigens in the context of primary and re-challenge infections were specific to conformational determinants. (B) IPs were performed on heated and unheated A2497 infected cell lysates using sera from monkey RML 134 and gels probed by Western blotting with chlamydial specific antibodies to identify heat sensitive and resistant conserved antigens. Heat treatment completely inhibited antibody reactivity against full length PmpD and its two proteolytically processed polypeptides and CPAF. Pgp3 and HSP60 antigenicity was partially affected or not affected by heating, respectively.</p