To validate the oxidative burst phenotype of Cyld+/- and Cyld-/- cells detected by APF- technique, we analyzed the ROS production by PECs using another independent method based on the chemiluminometric probe, luminol.
Our pilot experiments revealed that the most consistent results for luminol-based anylasis of oxidative burst could be generated using splenocytes. The use of PECs for oxidative burst assays was time and mice consuming. Only PECs with similar percentage e.g. of neutrophils and macrophages, after proteose peptone treatment of mice, could be subjected to the oxidative burst assay (see part 3.6.1.). Another problem is that pretreatment of mice with proteose peptone leads to differential priming level of PECs in each mouse, which would
result in non-consistent results in the subsequent oxidative burst assay. In contrast, splenocytes exhibit many advantages compared with PECs. (i) Splenocytes are removed from naïve mice which are not primed and thus should display similar background activities. (ii) Although the main ROS producers such as macrophages and granulocytes, , constitute together only ca. 10% of total splenocytes, this proportion is enough to detect ROS production using luminol, since the quantum yield of the luminol is very high and thus ROS production can be detected by using less than 100 phagocytes (Freitas et al., 2009).
Splenocytes obtained from Cyld+/- and Cyld-/- mice were infected with Y. enterocolitica WA(pYV) and real time production of ROS was determined by measuring chemiluminescence of luminol, using luminescence plate reader (see chapter 2.2.3.7.2). Our results indicated that Cyld-/--deficient splenocytes show higher respiratory oxidative burst response to Y. enterocolitica infection as measured by luminol-dependent chemiluminescence (relative light units: about 1,000 at the peak point) compared with that of Cyld+/- splenocytes (with the relative light units of about 500 at the peak point of stimulation) (Fig.26). Both of the Cyld+/- and Cyld-/- cells approached their respiratory oxidative burst peak quickly, around 10 to15 min p.i. (Fig.26). And then, the relative light units of Cyld+/- splenocytes dropped very quickly to the ground state (with relative light units of about 200) at 25 min while Cyld-/- cells did not approach to the basic level until ca. 45 min post infection (Fig.26).
In agreement with the results using APF, the analysis of ROS production during infection with L. monocytogenes and S. Typhimurium using luminol-dependent chemiluminescence showed similar level of respiratory burst response by Cyld+/- and Cyld-/- cells (Fig.27). Furthermore, in contrast to infection with Yersinia, Listeria infected splenocytes (both Cyld-/- and Cyld+/- cells) showed sustained high level of chemiluminescence (with the relative light units of about 1,000) during the whole analyzed infection time of ca. 85 min.
These data suggest that CYLD is involved in the negative regulation of cellular oxidative burst activity during Yersinia infection. Thus, we conclude that Yops-mediated inhibition of oxidative burst seems requires CYLD.
Fig. 26. ROS production by Cyld+/-and Cyld-/-splenocytes measured by luminol chemiluminescence. 105 splenocytes were seeded in 96-well plates in medium complemented with luminol. Cells were then infected with
Y. enterocolitica WA(pYV) at MOI = 10. Noninfected cells were used as control. The plate was centrifuged at 300 x g / 4°C / 2 min and the monitoring of luminescence was started immediately using luminescence plate reader. Each sample was analyzed in triplicate.
Fig. 27. ROS production by Cyld+/- and Cyld-/- splenocytes measured by luminol chemiluminescence. 105
splenocytes were seeded in 96-well plates in medium complemented with luminol. Cells were then infected with
L. monocytogenes at MOI = 19 (A) or S. Typhimurium(SB300 strain) at MOI = 44 (B). Noninfected cells were used as control. The plate was centrifuged at 300 x g / 4°C / 2 min and the monitoring of luminescence was started immediately using luminescence plate reader. Each sample was analyzed in triplicate.
Cyld+/+infected Cyld-/- infected Cyld-/- noninfected Cyld+/- noninfected Cyld+/+infected Cyld-/- infected Cyld-/- noninfected Cyld+/- noninfected A B
To analyze the direct contribution of CYLD to Yops-mediated inhibition of oxidative burst, we performed oxidative burst studies using different Y. enterocolitica mutants that are deficient in the expression of one yop gene. If CYLD is involved in Yop-mediated inhibition of oxidative burst, the infection of CYLD-deficient and CYLD-competent cells with strains deficient in this particular Yop should result in comparable oxidative burst responses.
It is known that YopH inhibits oxidative burst (Ruckdeschel et al., 1996). Furthermore, CYLD and YopP show overlapping functions e.g. as negative regulators of MAPK (Reiley et al., 2004; Yoshida et al., 2005; Zhou et al., 2005a). MAPKs are known to be involved in the activation of oxidative burst (Brown et al., 2004; El Benna et al., 1996a; El Benna et al., 1996b; Laroux et al., 2005; Sakamoto et al., 2006; Yamamori et al., 2002) and thus YopP is suggested to be also involved in the inhibition of ROS production by blocking the MAPK pathways (Visser et al., 1999).
Thus, we first concentrated on the analysis of oxidative burst response of Cyld-/- and Cyld+/- splenocytes to infection with WA(pYV∆YopH) and WA(pYV∆YopP).
Data presented in Figure.28 show that both mutant WA(pYV∆YopH) (Fig.28A) and WA(pYV∆YopP) (Fig.28B) stimulated ROS production of splenocytes and dropped to ground level of the respiratory oxidative burst much later (more than 50 min post stimulation) than that of infected Cyld+/- splenocytes (Fig.26). Thus, the stronger oxidative burst activity in Cyld-/- cells seems not to be a direct effect of CYLD, but rather due to the inability of YopH or YopP to inhibit oxidative burst in the absence of CYLD.
(A)
(B)
Fig. 28. ROS production by Cyld+/- and Cyld-/- splenocytes measured by luminol chemiluminescence. 105
splenocytes were seeded in 96-well plates in medium complemented with luminol. Cells were then infected with
mutant WA(pYV∆YopH at MOI = 27 (A) or with WA(pYV∆YopP) at MOI = 16 (B). Noninfected cells were
used as control. The plate was centrifuged at 300 g / 4°C / 2 min and the measurement of luminescence was
started immediately using luminescence plate reader. Each sample was analyzed in triplicate. Cyld-/-: blue line,
Cyld+/-: purple line.
Cyld-/-
Cyld+/-
Mutant WA(pYV∆YopH)
Cyld-/-
3.7 Analysis of cytokine production of infected Cyld
-/-and Cyld
+/-mice (in
vivo) and bone marrow derived dendritic cells (BMDCs, in vitro)
A major function of the innate immune cells during microbial infection is the production of inflammatory mediators such as cytokines, chemokines, nitric oxide, and ROS. The production of inflammatory cytokines is known to be critical for the resistance of the host to infections (Netea et al., 2004). The mobilization of the leukocytes to the infection site, the initiation of acute phase response and the adaptive immune response are some of the mechanisms triggered by proinflammatory cytokines and chemokines.
It has been shown that CYLDdeficiency results in enhanced induction of e.g. IL-1β, IL-6, IL- 8, IFN-α and IFN-β upon stimulation by agonists and/or infection (Yoshida et al., 2005; Zhang et al., 2006). Furthermore, Y. enterocolitica is known to inhibit cytokine production by immune cells (Brubaker, 2003; Monnazzi et al., 2004). Therefore, we wondered whether the improved resistance of Cyld-/- mice to Y. enterocolitica infection can be assigned to the enhanced cytokine response.