The impact of commonly used inhibitors of Cox-2 on viral infection and anti-viral immunity are largely unknown. We report that chronic administration of Cox-2 selective inhibitors blunts immune responses to VV. Antibody production, especially IgG2a, important for neutralizing virus, was severely attenuated in both Cox-2 deficient mice and mice chronically treated with Cox-2 selective inhibitors, although early acute treatment with Cox-2 inhibitors only had minor effects. IFN-γ production from activated spleen-derived CD4+ T cells was also severely impaired following treatment with SC-58125, indicating that T cell priming of antibody secreting cells could be at least partially responsible for attenuated antibody production. Further, chronic treatment with Cox-2 selective inhibitors resulted in elevated viral titers in vivo, indicating a loss of protective immunity. Overall, our results suggest that people that chronically use NSAIDs or Cox-2 selective drugs may not respond optimally to vaccination and could be more susceptible to viral infection.
Cox-2 selective inhibitors have the potential to dampen immune responses to vaccinations, autoimmune diseases and viral infections. Zhang et al.
demonstrated that B cells from a mouse lupus model expressed elevated levels of Cox-2 [22
]. Treatment of these mice with Celebrex, a Cox-2 selective inhibitor, decreased the production of autoantibodies and increased survival. Similarly, two models of adjuvant-induced arthritis showed that both Cox-2 deficient mice and rats treated with Cox-2 inhibitors had decreased autoantibody production [23
]. In conjunction with our data, these studies demonstrate that Cox-2 plays a critical role in production of antibody and elevated levels in autoimmune disease could contribute to disease progression. Further, Cox-2 selective inhibitors could be used as therapeutics to prevent or delay the onset of autoimmune diseases by reducing the production of pathogenic autoantibodies. Previously, we demonstrated that Cox-2 knockout mice vaccinated with non-infectious HPV-16 VLPs, used to prevent cervical cancer, had an attenuated humoral immune response [13
]. Although, the above findings suggest that humoral immune responses against self and to vaccines can be impaired in the absence of Cox-2, live virus infection was not evaluated. VV provides a well-studied and important model of infection, as well as being an efficacious vaccine against smallpox. Here, we showed that using a live VV infection, that Cox-2 deficient mice had a higher frequency of IgM producing cells and a lower frequency of IgG1, IgG2a and IgG3 producing cells. These data correlated with lower titers of VV neutralizing antibody in Cox-2−/−
mice. Thus, our new results demonstrate that the humoral immune response to live virus is impaired in mice that lack Cox-2 activity.
Cox-2 selective inhibitors are widely prescribed for chronic pain, arthritis, and even as supplements to cancer chemotherapy. As doses of Cox-2 inhibitors administered early in VV infection did not have a major impact on IgG antibody responses, we asked whether mice receiving chronic doses of Cox-2 selective inhibitors, similar to patient regimens, would have diminished humoral immunity. In our VV infected mice we report that neutralizing antibody titers were severely attenuated following chronic treatment with the Cox-2 selective inhibitor, SC-58125 ( & ). We observed decreased VV-specific IgM in SC-58125 treated mice on day 7 and day 21, but IgM levels recovered by day 28. These data matched well with results in Cox-2 deficient mice, VV-specific IgG titers and antibody secreting cell frequencies were also significantly attenuated in SC-58125 treated mice. IgG2a and IgG2b were the most highly affected isotypes, and these are the most important for viral neutralization. VV neutralizing titers were attenuated in mice administered Cox-2-selective inhibitors, most likely due to decreased IgG2a and IgG2b production. Subsequently, we observed higher viral titers in mice chronically treated with Cox-2 selective inhibitors. Our new results indicate that chronic administration of drugs that diminish Cox-2 activity could impair humoral immunity, making vaccines less efficacious and increasing susceptibility to viral infection.
We observed attenuated antibody production in mice chronically dosed with Cox-2 selective inhibitors and in Cox-2−/−
mice. This fully supports the concept that the effect of the Cox-2 selective drugs was Cox-2-dependent. High doses of drug can lead to off target effects [25
]. However, the doses we used 5 mg/kg, SC-58125, and 10 mg/kg, NS-398, fall within the range used in other studies and result in relatively low pharmacological concentrations [28
]. We observed some minor differences between Cox-2−/−
mice and mice dosed with Cox-2 selective inhibitors. For example, IgG2b levels were attenuated in Cox-2 inhibitor treated mice, but not in Cox-2−/−
mice. Although our results do not completely rule out Cox-2-independent effects using Cox-2 selective inhibitors, findings using Cox-2 knockout mice, suggest that few, if any, off target effects were responsible for the attenuated anti-viral humoral response.
Decreased IgG2a neutralizing antibody titers and antibody secreting cell frequencies prompted us to assess T cell responses within spleens of mice chronically treated with Cox-2-selective inhibitors. Production of IFN-γ by Th cells is important for optimal B cell activation and is necessary for class-switching in germinal centers, particularly to IgG2a and IgG2b [31
]. Mice lacking B cells, MHC class II or CD40 were more susceptible to secondary infection with a poxvirus, indicating that antibody production is essential for protection [33
]. Similarly, both B cell deficient and CD4 depleted mice failed to efficiently clear VV due to lack of neutralizing IgM and IgG antibodies and succumb to virus [16
]. Models of experimental autoimmune encephalomyelitis (EAE), a disease mediated by CD4+
Th1 cells, have shown that Cox-2 inhibitors prevent disease symptoms [35
]. Rofecoxib and Lumiracoxib, both Cox-2 inhibitors, were shown to reduce IFN-γ production from CD4+ T cells in an EAE model [36
]. By flow cytometric analysis we demonstrate the novel finding that VV infected mice treated with SC-58125 had reduced frequencies on day 7 of spleen-derived IFN-γ producing CD3+
cells. The CD4+
T cell response, essential for B cell neutralizing antibody production, peaks approximately 7 days post-infection with VV [14
]. We further demonstrated the attenuation of CD4+
IFN-γ producing cells in the presence of Cox-2 inhibitors by ELISPOT. These results suggest that reduced frequency of CD4+
T cells when Cox-2 is inhibited, at least partially accounts for the attenuated neutralizing antibody response directed against VV through reduced B cell class switching. We also observed a decrease in frequency of activated CD8+
T cells which could result in impairment of cellular immunity against VV, as well as protective memory against the virus, though memory was not investigated here.
Chronic treatment of mice with the Cox-2 selective inhibitor, SC-58125, resulted in reduced ability of splenocytes to produce prostaglandins ex vivo
. Previously, our lab demonstrated that PGE2
was critical for B cell class switching [38
]. This is consistent with our results showing that VV-specific IgM production was enhanced, particularly in Cox-2 deficient mice, while neutralizing IgG titers were reduced in both genetically deficient and mice treated with Cox-2 selective inhibitors, indicating a defect in class switch recombination. More recently, Yao et al.
reported that PGE2
promoted the differentiation of Th1 cells and their production of IFN-γ [39
]. The data presented herein, showing reduced frequencies of IFN-γ producing CD4 T cells are consistent with these findings. Taken as a whole, our data suggest that the decrease in Cox-2 activity and attenuated PGE2
production impaired both B and T cell function, which resulted in more severe viral infection.
Overall, our results demonstrate that chronic treatment with Cox-2 inhibitors attenuates humoral immunity generated against VV. These new data indicate that Cox-2 is essential for optimal in vivo
antibody production in response to viral infection. Chronic use of drugs that inhibit Cox-2, namely NSAIDs and Cox-2 selective inhibitors, could impair viral immunity, as well as vaccine generated immunity. If this were the case in humans, it would be particularly harmful to elderly patients, who commonly take NSAIDs and have difficulty achieving protective immunity to vaccination [40
]. Patients suffering from chronic pain, rheumatoid arthritis or osteoarthritis could also be more susceptible to infection and have reduced responses to vaccines, as they are often prescribed Cox-2 inhibitors. Clinical and epidemiological evaluations are necessary to determine whether this class of drugs will blunt human B cell antibody production following viral infection or vaccination. Our data herein clearly demonstrate that Cox-2-selective inhibitors diminish protective antibody responses to viral infection.