Bacterial lipopolysaccharide (endotoxin) triggers acute inflammation, associated with activation of the innate immune system and production of multiple proinflammatory mediators [19
]. In this study, we attempted to utilize the proinflammatory capability of LPS to enhance the incidence of the naturally occurring inflammatory joint disease ANKENT in mice.
ANKENT develops almost exclusively in males and this hallmark of the disease is associated with hormonal and behavioural factors – similarly as in naturally occurring arthritis in DBA/1 male mice [21
]. The males caged together have a significantly higher risk of ANKENT development than males caged alone [22
]. During the whole experiment both LPS-treated and LPS-untreated males were caged under the same conditions and we have not observed any significant difference in behaviour between the groups.
Previously, we showed that germ-free mice do not suffer from the joint disease [13
]. However, after their colonization with a mixture containing common intestinal bacteria, some mice developed ANKENT [14
]. The close connection between the gut and SpA was repeatedly demonstrated in laboratory animals [23
] and was also confirmed in humans [28
]. Because LPS is an agent which when administered intraperitoneally enhances intestinal permeability, and leads to translocation of commensal microbiota from the gut [17
], we expected a higher frequency of the disease in LPS-treated males. Surprisingly, LPS administration to ANKENT-susceptible B10.BR male mice in their early adulthood acted protectively: the incidence of ANKENT in LPS-treated males was significantly reduced compared to control LPS-untreated group. This unexpected result could be explained by an overactivation of innate immunity cells leading to an upregulation of negative regulatory pathways [34
The second aim of our work was to study the immune response. The cellular infiltrate in joints and elevated IL-6 levels in the sera were demonstrated in ANKENT positive animals at the time when ANKENT became manifest [9
]. These signs disappeared after the period of acute inflammation. In this study we did not test mice in the acute stages of the disease, but we were interested in the immune response that precedes ANKENT onset. We compared populations of immune cells and cytokine levels in LPS-treated group, where ANKENT occurrence was suppressed, with the control LPS-untreated group, where ANKENT was manifest.
Throughout the course of LPS administration, significantly higher percentages of macrophages (CD11b+), dendritic cells (CD11c+), neutrophils (Ly6G+) and also natural killer cells (CD3-CD49b+) were found in LPS-treated males than in LPS-untreated ones. This LPS-induced expansion of innate immune cells in spleen was transient and was not present at the end of experiment when the frequencies of these populations were similar in both groups.
Just the opposite effect of LPS treatment was observed in lymphocyte subpopulations. During LPS administration the percentage of B cells, T cells and T helper cells in the spleen was significantly decreased in LPS-treated males and the decrease in frequency of B cells lasted throughout LPS treatment. At the end of the experiment, no differences were found between LPS-treated and LPS-untreated males. This suggests that cells of the adaptive immune system did not directly participate in the immune response to LPS treatment, and the suppression of adaptive immunity in LPS-treated males might be involved in the reduction of ANKENT incidence.
Specific cells of innate and adaptive immunity are associated with the production of cytokines. In LPS-treated males we found significantly increased serum levels of IL-6 and IL-10 during LPS administration, whereas the percentage of CD4+ T cells, which is an important source of these cytokines, was decreased. However, macrophages also produce IL-6 and monocytes, whose frequency was strongly elevated in LPS-treated males, are the main cellular source of IL-10.
The finding of significantly elevated IL-10 levels in sera of LPS treated males was discrepant with the in vitro
response of LPS stimulated splenocytes, which did not secrete detectable IL-10. This contradiction between serum and in vitro
spleen cell response to LPS stimulation could be explained by the fact that the major source of circulating IL-10 after LPS stimulation is liver [35
No significant serum differences were found between LPS-treated and LPS-untreated males in IL-6 and IL-10 and in all other tested cytokines at the end of the experiment. However, IL-6 and TNFα were elevated in both groups. The elevation of these pro-inflammatory cytokines at the end of experiment could be explained by the presence of ANKENT-positive animals. We could only speculate that LPS treatment suppressed and delayed the onset of ANKENT and that cytokine data both from serum and from in vitro stimulation show the subclinical signs of disease. Nevertheless, at the end of the experiment we have detected only a single affected mouse in LPS-treated group. To evaluate the data in more detail we would need to match single cytokine data with individual mice. However, both LPS-treated as well as LPS-untreated control males were evaluated as a group.
We do not know the exact mechanism of LPS effect in ANKENT development, but the high level of anti-inflammatory cytokine IL-10 could contribute to homeostasis in LPS-treated males. IL-10, which is a potent anti-inflammatory agent, might suppress the onset of ANKENT by its effect on the secretion of pro-inflammatory cytokines including TNFα and IL-6. A regulation of IL-10 is connected with a function of pro-inflammatory TNFα. In mice, TNFα up-regulates LPS-induced IL-10 synthesis and neutralization of TNFα with anti TNFα antibody results in a significant reduction of LPS-inducible IL-10 production [35
To better understand the immune mechanisms underlying the development of ANKENT, in vitro
LPS stimulation of splenocytes was performed. In the course of LPS administration we observed an elevated level of IL-6 in supernatants of splenocytes from LPS-treated males. This result was consistent with the elevated IL-6 levels in blood sera from the LPS group. At the end of the experiment we found not only further increase in IL-6 levels but also increase in TNFα levels in both groups. The levels of both TNFα and IL-6 were significantly higher in LPS-treated group compared to control group. TNFα is directly produced by macrophages in response to LPS stimulation [36
] and is also a typical marker for AS in man [37
]. The anti-TNF monoclonal antibodies that act by neutralizing TNF have proved highly effective in AS therapy, but also in the treatment of other spondyloarthropathies [38