Anti-Tumor Necrosis Factor α Therapeutics Differentially Affect Leishmania Infection of Human Macrophages

Abstract

Tumor necrosis factor α (TNFα) drives the pathophysiology of human autoimmune diseases and consequently, neutralizing antibodies (Abs) or Ab-derived molecules directed against TNFα are essential therapeutics. As treatment with several TNFα blockers has been reported to entail a higher risk of infectious diseases such as leishmaniasis, we established an in vitro model based on Leishmania-infected human macrophages, co-cultured with autologous T-cells, for the analysis and comparison of anti-TNFα therapeutics. We demonstrate that neutralization of soluble TNFα (sTNFα) by the anti-TNFα Abs Humira^®, Remicade^®, and its biosimilar Remsima^® negatively affects infection as treatment with these agents significantly reduces Leishmania-induced T-cell proliferation and increases the number of infected macrophages. By contrast, we show that blockade of sTNFα by Cimzia^® does not affect T-cell proliferation and infection rates. Moreover, compared to Remicade^®, treatment with Cimzia^® does not impair the expression of cytolytic effector proteins in proliferating T-cells. Our data demonstrate that Cimzia^® supports parasite control through its conjugated polyethylene glycol (PEG) moiety as PEGylation of Remicade^® improves the clearance of intracellular Leishmania. This effect can be linked to complement activation, with levels of complement component C5a being increased upon treatment with Cimzia^® or a PEGylated form of Remicade^®. Taken together, we provide an in vitro model of human leishmaniasis that allows direct comparison of different anti-TNFα agents. Our results enhance the understanding of the efficacy and adverse effects of TNFα blockers and they contribute to evaluate anti-TNFα therapy for patients living in countries with a high prevalence of leishmaniasis.

Keywords: T-cells; cimzia®; complement; human macrophages; leishmaniasis; polyethylene glycol; remicade®; tumor necrosis factor α.

Figure 1

Expression of tumor necrosis factor α (TNFα) and mTNFRs by human monocyte-derived macrophages (hMDMs) or T-cells after Leishmania major (Lm) infection. Macrophages were infected with fluorescent Lm (multiplicity of infection = 20) and analyzed after 24 h. (A) Infection rates in hMDMs were determined by flow cytometry in comparison to non-infected hMDMs. (B) Infected and non-infected hMDMs were stained with Diff-Quik^® and representative micrographs are depicted. Black arrows indicate Lm parasites. (C) The concentration of soluble TNFα (sTNFα) in supernatants of hMDMs in the presence or absence of Lm was measured by ELISA 24 h post-infection. (D) Cell surface expression of mTNFR1, mTNFR2, or mTNFα on hMDMs was analyzed by flow cytometry 24 h after infection. (E) Peripheral blood lymphocytes were co-incubated with infected/non-infected hMDMs and cells were measured by flow cytometry after 7 days, with T-cells being defined by anti-CD3 Ab co-staining. (D,E) Relative fluorescence intensity was measured as the ratio of the mean fluorescence intensity (MFI) of specific markers to the MFI of isotype controls. Histograms show expression on hMDMs or T-cells in the non-infected (gray line) or infected culture (black line) in comparison to the isotype controls (black/gray solid). Data are presented as mean ± SD (n ≥ 7) and were obtained from at least two independent experiments. The Wilcoxon signed-rank test was performed to evaluate statistical significance. *P < 0.05.

Figure 2

Proliferation of CD4⁺ T-cells reduces the number of Leishmania major (Lm)-infected macrophages. (A,B) Lm-infected/non-infected human monocyte-derived macrophages (hMDMs) were incubated in the absence or presence of CFSE-labeled autologous peripheral blood lymphocytes (PBLs). 7 days post-infection, T-cell proliferation (−Lm , +Lm ●) and infection rates (−PBLs , +PBLs ●) were measured by flow cytometry with T-cells being defined by anti-CD3 Ab co-staining. T-cell proliferation (A) and infection rates (B) are illustrated as representative histograms (upper panels), raw data (middle panels), or as differences by subtracting the respective controls from each corresponding donor (lower panels). (C) CD4/CD8 subset distribution was determined for proliferating (CFSE^low) and non-proliferating (CFSE^high) CD3⁺ T-cells in Lm-infected/non-infected hMDM/PBL co-cultures after 7 days. At least three independent experiments were conducted of which data are presented as mean ± SD (n ≥ 8). To determine statistical significance, the Wilcoxon signed-rank test was performed. *P < 0.05.

Figure 3

TNFα blockers have diverging effects on T-cell proliferation and Lm infection rates. (A) Schematic representation of the TNFα blockers Remicade^® (Re), Remsima^® (Rs), Humira^® (Hu), and Cimzia^® (Ci). (B–D) Lm (MOI = 20) were added to hMDMs for 3 h. Subsequently, extracellular parasites were removed by washing. Anti-TNFα agents (Re ○, Rs □, Hu Δ and Ci ◊) were added 24 h after Lm infection (−Lm , +Lm ●) of hMDMs and incubated in the presence of CFSE-labeled autologous PBLs. (B) The concentration of sTNFα was measured by ELISA 7 days post-infection. T-cell proliferation (C) and Lm infection rates in hMDMs (D) were analyzed by flow cytometry after 7 days. (C,D) Values of the corresponding controls were subtracted for each donor and condition to illustrate differences. Results are presented as mean ± SD (n ≥ 7) and at least four separate experiments were conducted. The Wilcoxon signed-rank test was performed to evaluate statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: CDRs, complementarity determining regions; Fv, fragment variable; TNFα, tumor necrosis factor α; Lm, Leishmania major; MOI, multiplicity of infection; hMDMs, human monocyte- derived macrophages; PBLs, peripheral blood lymphocytes; sTNFα, soluble TNFα.

Figure 4

Effects of anti-tumor necrosis factor α (TNFα) agents are human monocyte-derived macrophage (hMDM)- and T-cell-mediated. Magnetic activated cell sorting-separated CD14⁺ hMDMs were infected with Leishmania major (Lm) (−Lm , +Lm ●) and co-cultured with untouched CD3⁺ T-cells. The anti-TNFα agents Remicade^® (Re ○) and Cimzia^® (Ci ◊) were added and after 7 days, T-cell proliferation (A,B) and Lm infection rates in hMDMs (C,D) were analyzed by flow cytometry. (A,C) Raw data of T-cell proliferation and Lm infection rates are depicted without subtracting values from controls. (B,D) Values of the respective controls were subtracted for each donor and condition to illustrate differences. Results are presented as mean ± SD (n ≥ 9) and at least four separate experiments were conducted. The Wilcoxon signed-rank test was performed to evaluate statistical significance. ns P > 0.05; **P < 0.01.

Figure 5

Intracellular expression of cytolytic proteins in proliferating CD4⁺ T-cells differs after soluble TNFα (sTNFα) neutralization by Remicade^® or Cimzia^®. Non-infected () or Leishmania major (Lm)-infected human monocyte-derived macrophages (hMDMs) (●) were co-incubated with CFSE-labeled autologous peripheral blood lymphocytes and sTNFα was neutralized by Remicade^® (Re ○) or Cimzia^® (Ci ◊). Proliferating CD4⁺ T-cells expressing perforin (A), granulysin (B), granzyme A (C), or granzyme B (D) were detected using anti-CD3 and anti-CD4 Ab co-staining in intracellular flow cytometry 7 days post-infection. Values are illustrated as differences to the untreated control of the same donor. Data are presented as mean ± SD (n ≥ 6) and were obtained in at least three independent experiments. To analyze statistical significance, the Wilcoxon signed-rank test was performed. *P < 0.05; **P < 0.01.

Figure 6

Reduced T-cell proliferation and increased Leishmania major (Lm) infection rates upon soluble TNFα (sTNFα) blockade by Remicade^® are independent of fragment crystallizable (Fc)–Fcγ receptor (FcγR) interactions. (A) Representative histograms of flow cytometry analysis show FcγR expression (black line) on infected human monocyte-derived macrophages (hMDMs) in comparison to the isotype control (black solid) 24 h after Lm infection. (B) 7 days after infection, binding of Polyglobin^® Abs to FcγRs on Lm-infected and Polyglobin^®-treated hMDMs (black dotted line) in comparison to non-treated cells (black solid) was confirmed by flow cytometry using anti-human IgG staining. (C,D) Lm-infected (●) or non-infected () macrophages were pre-incubated with or without Polyglobin^® after which peripheral blood lymphocytes and Remicade^® (Re) were added (Remicade^® ○; Polyglobin^® ×; Remicade^® + Polyglobin^® ⊗). (C) T-cell proliferation and (D) infection rates in hMDMs of co-cultures were assessed by flow cytometry 7 days post-infection. Values are illustrated as differences to the untreated control of the same donor. Results are presented as mean ± SD (n ≥ 8) and were obtained from at least three independent experiments. Statistical analysis was carried out using the Wilcoxon signed-rank test. ns P > 0.05.

Figure 7

Increased T-cell proliferation and reduced Leishmania major (Lm) infection rates in human monocyte-derived macrophages (hMDMs) after PEGylation of Remicade^®. The Lm-infected hMDM/peripheral blood lymphocyte co-culture (●) was treated with Remicade^® (○), PEGylated Remicade^® (), Cimzia^® (◊), and MS-polyethylene glycol (PEG) (∗). 7 days after infection, soluble TNFα (sTNFα) neutralization (A) or C5a levels (D) were measured by ELISA, and T-cell proliferation (B) as well as the percentage of Lm-infected hMDMs (C) were determined by flow cytometry. (B,C) T-cell proliferation and infection rates are presented as differences to the untreated control of the same donor. At least four independent experiments were conducted of which data are shown as mean ± SD (n ≥ 10). The Wilcoxon signed-rank test was performed to evaluate statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.