Cladribine Exposure Results in a Sustained Modulation of the Cytokine Response in Human Peripheral Blood Mononuclear Cells

<div><p>Background and Objectives</p><p>Cladribine is a cytotoxic drug which ameliorates the clinical course of relapsing-remitting multiple sclerosis. In addition to cytotoxicity, the mode of action may include immunomodulatory mechanisms. This <i>in vitro</i> study was designed to investigate cladribine’s effects on cell function after the removal of cladribine to distinguish cytotoxic versus immunomodulatory effects.</p><p>Methods</p><p>Cells were incubated in the absence or presence of cladribine (1×10^-8 M to 1×10^-5 M) for 72 h. Cladribine was removed from the cell culture and surviving peripheral blood mononuclear cells were cultured up to 58 days to determine the immunomodulatory effects of cladribine on cell function (e.g., proliferation and cytokine release).</p><p>Results</p><p>In the long-term, brief cladribine exposure did not impair the proliferation of surviving peripheral blood mononuclear cells. However, it induced an anti-inflammatory shift in the cytokine milieu with significantly enhanced release of IL-4 (Days 9 and 44, p<0.01; Day 58, p<0.05) and IL-5 (Day 9, p<0.01), resulting in an increased IL-4/INF-gamma ratio (Days 9 and 44, p<0.01; Day 58, p<0.05). Additionally, a trend towards an increased IL-10 production was observed. No changes were found in the production of IFN-gamma, TNF-alpha, IL-6, IL-8, IL-17A, IL-23 or NGF-beta.</p><p>Conclusions</p><p><i>In vitro</i> cladribine exposure induces a sustained anti-inflammatory shift in the cytokine profile of surviving peripheral blood mononuclear cells. This immunomodulatory action might contribute to cladribine’s beneficial effects in the treatment of multiple sclerosis.</p></div

Treg depletion prior to immunization of NSI-treated mice.

<p>DEREG mice (n = 12) were non-specifically immunized (NSI, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192197#pone.0192197.ref029" target="_blank">29</a>]). On days 5 and 6, the animals were given 1 μg of diphtheria toxin intraperitoneally to deplete Tregs; control mice received the same volume of PBS (n = 12). Seven days after NSI, the animals were primed by intra-peritoneal immunization with TNP-OVA/OVA in alum. Animals without NSI and immunization (n = 6) and animals immunized with TNP-OVA/OVA without prior NSI (n = 9) served as controls. The relative concentration of anti-OVA IgG (A) and the relative antibody affinity (B) were measured as described in the caption of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192197#pone.0192197.g005" target="_blank">Fig 5</a>. (C) Numbers of OVA-specific IgG-secreting cells in the bone marrow were determined in an ELISpot assay. (D) The proliferative response of splenocytes to restimulation with OVA was measured in a proliferation assay <i>ex vivo</i>. (E) The proliferative response to concanavalin A (ConA) served as control. Means and the 95% confidence interval are shown. Group differences were tested for significance with the Kruskal-Wallis test and Dunn’s multiple comparison for selected pairs. Results from two separate experiments were combined. *** P <0.001; ** P <0.01; * P <0.05.</p

Experimental Sepsis Impairs Humoral Memory in Mice

<div><p>Patients with sepsis are often immune suppressed, and experimental mouse models of sepsis also display this feature. However, acute sepsis in mice is also characterized by a generalized B cell activation and plasma cell differentiation, resulting in a marked increase in serum antibody concentration. Its effects on humoral memory are not clearly defined. We measured the effects of experimental sepsis on long-term immunological memory for a defined antigen: we induced colon ascendens stent peritonitis (CASP) 8 weeks after 2 rounds of immunization with ovalbumin. Four weeks later, the antigen-specific bone marrow plasma cell count had doubled in immunized non-septic animals, but remained unchanged in immunized septic animals. Sepsis also caused a decrease in antigen-specific serum antibody concentration. We conclude that sepsis weakens humoral memory by impeding the antigen-specific plasma cell pool’s development, which is not complete 8 weeks after secondary immunization.</p> </div

Suppression of the adaptive immune response after sepsis induction.

<p>Sepsis was induced in C57BL/6 mice, and on day 7, the animals were immunized intraperitoneally with TNP-OVA/OVA in alum (n = 47). Non-septic, not immunized animals (n = 32) and non-septic immunized animals (n = 25) served as controls. The antigen-specific immune response was determined 14 days after the immunization by measuring the serum concentration of anti-OVA IgG by ELISA. (A) The relative concentrations of OVA-specific IgG antibodies in serum normalized to a standard serum are shown (arbitrary units, AU). (B) The proliferative response to restimulation with OVA was measured by thymidine incorporation <i>ex vivo</i>. (C) The proliferative response to the mitogen concanavalin A (ConA) served as control. (D) Animals that had been immunized post-sepsis were divided into two groups: severely immunosuppressed mice (n = 17) had OVA-specific IgG concentrations below 10% of control values, while mildly immunosuppressed animals (n = 30) had serum concentrations greater than or equal to 10%. (E) Numbers of OVA-specific IgG-secreting cells in the bone marrow were determined in an ELISpot assay. (F) The relative antibody affinity of TNP-specific IgG was measured as binding to slightly haptenized TNP-BSA in comparison to highly haptenized TNP-BSA in an ELISA (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192197#pone.0192197.ref034" target="_blank">34</a>]). (G-J) Blood was drawn 24 h after sepsis induction and cytokine concentrations of IL-6 (G), IL-10 (H), MCP-1 (I), and TNF (J) were quantified in the sera. Severely (n = 15) and mildly immunosuppressed (n = 29) animals are compared with non-septic, not immunized controls (n = 12). In the panels A-F means and 95% confidence intervals are shown. In the panels G-J box plots depict medians and quartiles. Group differences were tested for significance with the Kruskal-Wallis test and Dunn’s multiple comparison for selected pairs. Results were pooled from five separate experiments. *** P <0.001; ** P <0.01; * P <0.05.</p

Treg depletion prior to immunization of post-septic animals.

<p>In DEREG mice (n = 15) and C57BL/6 wild-type controls (n = 9) sepsis was induced, and on days 5 and 6 the animals were given 1 μg of diphtheria toxin intraperitoneally to deplete Tregs. On day 7, the animals were immunized intraperitoneally with TNP-OVA/OVA in alum. Non-septic, not immunized animals (n = 11) and non-septic immunized animals (n = 10) served as controls. (A) 14 days after immunization, the antigen-specific immune response was determined by the concentration of anti-OVA IgG in the serum via ELISA and normalized to a standard serum (arbitrary units, AU). (B) The proliferative response of splenocytes to restimulation with OVA was measured by thymidine incorporation <i>ex vivo</i>. (C) The proliferative response to the mitogen concanavalin A (ConA) served as control. Means and 95% confidence intervals are shown. Group differences were tested for significance with the Kruskal-Wallis test and Dunn’s multiple comparison for selected pairs. Pooled results from three separate experiments with the same tendency are shown. *** P <0.001; ** P <0.01; * P <0.05.</p

Suppression of the adaptive immune response after non-specific immunization.

<p>(A, B) NSI alone did not induce OVA-specific serum antibodies but increased total serum IgG. OVA-specific IgG (A) and total serum IgG (B) were measured 21 days after NSI (n = 3). Animals without NSI and immunization (n = 3 and 6), only immunized animals (n = 5 and 11), and animals immunized 7 days after NSI (n = 6 and 11) served as controls. (C, D) NSI reduced the specific response to antigen priming. C57BL/6 mice were non-specifically immunized (NSI, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192197#pone.0192197.ref029" target="_blank">29</a>]). At the indicated time points after NSI, the animals were primed intraperitoneally with TNP-OVA/OVA in alum (n = 5–6). Animals without NSI and immunization (n = 6) and animals immunized without prior NSI (n = 5) served as controls. The antigen-specific immune response was measured as the concentration of anti-OVA IgG in the serum 14 days after priming with OVA by an ELISA. (C) The relative concentrations of OVA-specific IgG antibodies in serum normalized to a standard serum (arbitrary unit, AU) are shown. (D) The relative antibody affinity of TNP-specific IgG was measured as binding to slightly haptenized TNP-BSA in comparison to highly haptenized TNP-BSA in an ELISA (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192197#pone.0192197.ref034" target="_blank">34</a>]). (E) The NSI-induced immune suppression was independent of CpG. C57BL/6 mice were treated with NSI containing CpG (NSI, n = 12) or not (NSI w/o CpG, n = 6). Seven days later, they were immunized intraperitoneally with TNP-OVA/OVA in alum (OVA). Mice without NSI and immunization (n = 7) and animals immunized only (n = 9) served as controls. The antigen-specific immune response was determined as described for panel C. In the panels A-D means and 95% confidence intervals are shown. Kruskal-Wallis test and Dunn’s multiple comparison for selected pairs. In panels B and E pooled results from two separate experiments are shown. *** P <0.001; * P <0.05.</p

<i>In vitro</i> effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23 and 30 after transfer into cladribine-free medium.

<p><b>PBMCs from healthy blood donors (n = 4) were incubated in the absence or presence of cladribine</b>. (1×10⁻⁸ M to 5×10⁻⁷ M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with PHA for 48 h at days 9, 16, 23 and 30; supernatants were collected and cytokine were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.</p

Experimental sepsis in OVA-immunized mice reduces OVA-specific IgG-ASCs.

<div><p>OVA-specific IgG-ASCs in spleen and bone marrow 2 and 4 wk after CASP (colon ascendens stent peritonitis) are shown here. C57BL/6 mice were immunized and boosted with OVA at 6 and 9 wk, respectively, and CASP was induced 8 wk after the secondary immunization. Control animals were either untreated, only immunized or only CASP induced. A combination of two independent experiments with the same trend is shown here. A statistical analysis was performed with the one-way ANOVA and Bonferroni’s post test for selected pairs. N = 10-18/group. </p> <p>*: p<0.05; **: p<0.01. </p></div

Experimental sepsis in OVA-immunized mice reduces OVA-specific serum IgG.

<div><p>OVA-specific serum-IgM and -IgG at 2 and 4 wk after CASP (colon ascendens stent peritonitis) are shown here (au = arbitrary units). C57BL/6 mice were immunized and boosted with OVA at 6 and 9 wk, respectively, and CASP was induced 8 wk after the secondary immunization. Control animals were either untreated, only immunized or only CASP induced. A combination of two independent experiments with the same trend is shown here. A statistical analysis was performed with the one-way ANOVA and Bonferroni’s post test for selected pairs. </p> <p>n=10-18/group. *: p<0.05; **: p<0.01.</p></div

<i>In vitro</i> effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23, 30, 44 and 58 after transfer into cladribine-free medium.

<p>PBMCs from healthy blood donors (n = 5) were incubated in the absence or presence of cladribine. (1×10⁻⁸ M to 5×10⁻⁷ M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with anti-CD3/anti-CD28 antibodies for 48 h at days 9, 16, 23, 30, 44 and 58; supernatants were collected and cytokine concentrations were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.</p