Interleukin-11 receptor expression on monocytes is dispensable for their recruitment and pathogen uptake during Leishmania major infection.

Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.

Introduction

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines [1]. It activates its target cells by first binding to the membrane-bound IL-11 receptor (IL-11R), which subsequently results in the recruitment of two glycoprotein 130 (gp130) molecules, the formation of a gp130 homodimer and the activation of intracellular signaling cascades, most notably the Jak/STAT signaling pathway [2]. Furthermore, IL-11 can bind to soluble forms of the IL-11R (sIL-11R), which also induce gp130 homodimerisation and activate intracellular signaling [3]. Although the biological functions of IL-11 are under intense investigation, it is established that IL-11 has important immunomodulatory functions by acting on macrophages/monocytes, CD4+ T cells and B cells [4]. Inhibition of IL-11 signaling, e.g. by monoclonal antibodies, has been proposed as a therapeutic strategy for a number of diseases, including cancer, cardiovascular and lung diseases [5].

The eukaryotic parasite Leishmania is the causative agent of a variety of cutaneous, mucocutaneous and visceral infections termed leishmaniasis. One important representative is Leishmania major (L. major), which infects phagocytes of its hosts and not only escapes the innate defense mechanisms of the endocytic compartments, but is rather able to survive and replicate within the phagocytes [6]. This ability to prevent eradication by the immune system of the host often results in chronic infections, causing distress for the patient. Although different immune cells, including neutrophils, monocytes, macrophages and dendritic cells are reservoirs of L. major in the early phase of the infection [7], it has been shown that especially monocyte-derived cells play an important role in inducing protective immune responses, but also act as a niche for the parasite [8], [9], [10].

Recently, increased levels of IL-11 mRNA in the lesions of L. donovani-infected patients have been reported [11]. However, a role for IL-11 on the monocyte recruitment and pathogen uptake during Leishmania infection has not been investigated so far. In this study, we show that primary monocytes express IL-11R on their cell surface. Furthermore, we analyze the contribution of IL-11 signaling on monocyte recruitment and infection by L. major in a mouse model of cutaneous leishmaniasis.

Materials and methods

Ethics statement

All animal experiments were reviewed and approved by the Ethics Committee of the Office for Veterinary Affairs of the State of Saxony-Anhalt, Germany (permit license numbers IMKI/G/01-1314/15 and IMKI/G/01-1575/19) in accordance with legislation of both the European Union (Council Directive 499 2010/63/EU) and the Federal Republic of Germany (according to § 8, Section 1 TierSchG, and TierSchVersV). Ethic approval for the experiment with primary human monocytes was obtained from the review board of the medical faculty of Kiel University (study #D 487/13). All participants gave written informed consent. Peripheral blood from healthy volunteers was collected by venipuncture.

Parasites and mouse infections

L. major LRC-L137 V121 DsRed parasites were previously described [9]. Parasites were grown in M119 medium completed with 10% heat-inactivated fetal calf serum, 0.1 mM adenine, 1 mg/ml biotin, 5 mg/ml hemin, and 2 mg/ml biopterin (all from Sigma) for maximally 6 passages. For the infection of mice, stationary phase parasites were centrifuged (600 g, 5 min, RT) and resuspended in PBS. 2 × 106 parasites in 10 μl PBS were subsequently injected into the ear dermis. Analysis was performed 3 weeks post infection. All mice were bred and housed under specific pathogen-free conditions in the central animal facility (ZTL) of the Medical Faculty at Otto-von-Guericke-Magdeburg.

Adoptive cell transfer

Mice deficient for IL-11R have been described previously [12]. For bone marrow isolation, bone marrow cells from CD45.1+ wild type, CD45.2+/IL-11R+/+ and CD45.2+/IL-11R−/− mice were flushed out of tibia and femur with ice cold non-supplemented RMPI medium and filtered through 100 µm cell strainers. CD45.1+ WT and CD45.2+ IL-11R−/− cells were mixed in a 1:1 ratio, labeled with Carboxyfluorescein succinimidyl ester (CFSE) for 20 min at 37 °C in non-supplemented PBS, washed with PBS supplemented with 10% FCS, and 8 × 107 cells per recipient were resuspended in PBS and injected intravenously into the tail vein of CD45.2+ C57BL/6 mice. As a control for IL-11R deficiency, CFSE-labelled CD45.1+ WT and CD45.2+ IL-11R+/+ cells were injected intravenously into the tail vein of CD45.2+ C57BL/6 mice. Five days post transfer, immune cells isolated from the infected ears were analyzed via flow cytometry.

Flow cytometry

Human blood was collected in heparin vials and blocked with 1:100 human serum and human TruStain FcX (BioLegend, San Diego, CA, USA) before staining. IL-11R was stained with the anti-IL-11R antibody N-20 (sc-993, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA) and AlexaFluor488-conjugated goat anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA). Monocytes were stained using PE anti-human CD14 (clone M5E2) (Biolegend). After staining, erythrocytes were lysed and cells were fixed using RBC Lysis/Fixation Solution (BioLegend) and measured with a Canto II flow cytometer (BD Biosciences, Heidelberg, Germany).

Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 0.1 mg/ml Liberase™ TL (Sigma-Aldrich) and 4 µg/ml DNase (Sigma-Aldrich) for 60 min shaking at 600 rpm and 37 °C, and passed through a 70 μm cell strainer. Surface staining of cells was done by using APC conjugated anti-CD11b (clone M1/70), APC-Cy7 conjugated anti-CD45.1 (clone A20), PE-Cy7 conjugated anti-CD45.2 (clone 104), BV421 conjugated anti-Ly6G (clone 1A8), BV510 conjugated anti-MHC class II (IA/IE, clone M5/114.15.2) and BV785 conjugated anti-Ly6C (clone HK1.4), which were all purchased from BioLegend. DsRed fluorescence was read out at 558 nm excitation and 585/15 nm emission. An autofluorescence signal was recorded at 488 excitation and 695/40 nm emission. Samples were Fc-blocked using anti-CD16/32 antibody (clone 93) (BioLegend) before antibody staining. Analysis was performed with a Fortessa or FACS ARIA III (BD Biosciences) using 405, 488, 561, and 633 nm lasers. Data were analyzed by using the FlowJo X software (FlowJo, LLC). Relative MFI of IL-11R staining was calculated by subtraction of unstained control.

Statistical analysis

Statistical analysis was carried out with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data from infection experiments were analyzed by a Two-way ANOVA (grouped into individual experiments, comparing the effect of the genotype) and a Bonferroni post test for differences between experimental conditions.

Results

IL-11 receptor is expressed on human monocytes

The expression pattern of the IL-11R determines which cell types can be activated by IL-11. Although the cytokine is known for more than 30 years, only little knowledge exists about its functions on immune cells and thus their IL-11R expression. We have previously shown high IL-11R expression on human primary CD66b+ polymorphonuclear leukocytes [3], but a comprehensive analysis is lacking. In order to elucidate a potential role of IL-11 signaling in monocytes during Leishmania major infection, we first determined whether IL-11R was expressed on monocytes. We were able to successfully show the expression of IL-11R on the human monocytic cell line THP-1 (Fig. 1A), which is in good agreement with previous results [3], [13]. We substantiated this by IL-11R staining on CD14+ cells from whole human blood of healthy volunteers. IL-11R expression was variable among individuals, but clearly detectable in all subjects (Fig. 1B, C). Importantly, almost all cells positive for CD14 were also positive for IL-11R, and this did not vary substantially between individuals (Fig. 1D). Collectively, these data show that IL-11R is expressed on monocytic cell lines as well as primary human monocytes.

Fig. 1

Monocytes express IL-11R on their cell surface. (A) IL-11R expression on the monocytic cell line THP-1 was determined using flow cytometry (blue curve). THP-1 cells exclusively stained with the secondary antibody were used as control (filled gray curve). (B, C) Blood was obtained from five healthy donors and IL-11R expression on CD14+ cells was determined via flow cytometry. Representative plots compared to an unstained control (gray) are shown in panel (B). The relative mean fluorescence intensity of the five samples is shown in panel (C). (D) Percentage of CD14/IL-11R double positive cells from the five healthy donors. MFI, mean fluorescence intensity.

Ablation of IL-11R signaling does not influence Leishmania infection in monocytes in vivo

Having shown that the IL-11R is expressed on monocytes, we sought to investigate a possible impact on monocytes during L. major infection. We used an adoptive transfer approach in order to compare wild type and IL-11R-deficient monocytes side by side [9]. For this, we injected 2 × 106 DsRed-labelled Leishmania major parasites intradermally into the ear of C57BL/6 mice. 16 days post infection, we injected a 1:1 mixture of either CFSE-labelled CD45.1+ WT cells and CD45.2+ IL-11R−/− or, as a control for IL-11R deficiency, CFSE-labelled CD45.1+ WT cells and CD45.2+ IL-11R+/+ intravenously into the tail vein of the mice (Fig. 2A). Five days after cell transfer, we analyzed the immune cells isolated from the infected ears via flow cytometry. First, we determined whether the IL-11R affects monocyte recruitment. For this, we determined for the CFSE-labeled, recruited cell population the ratio between CD45.1+ and CD45.2+ cells for the mice which had received CD45.2+ cells either from IL-11R+/+ or IL-11R−/− donors together with CD45.1+ control cells (Fig. 2B). We observed no significant difference in the recruitment of IL-11R+/+ versus IL-11R−/− cells (Fig. 2C). In order to analyze the proportion of IL-11R−/− cells among different monocyte populations, we analyzed Ly6C+ MHCII- (recently recruited), Ly6C+ MHCII+ (intermediate) and Ly6C- MHCII+ (matured) monocytes separately (Fig. 2D), yielding no significant differences (Fig. 2E). Therefore, these data exclude a major role for IL-11R signaling on monocyte recruitment to the site of infection during ongoing L. major infection.

Fig. 2

IL-11R does not influence monocyte recruitment or infection. (A) Experimental strategy to determine monocyte recruitment and infection of CFSE-labelled, newly recruited IL-11R+/+ or IL-11R−/− (CD45.2+) and WT control (CD45.1+) cells side-by-side. (B) Gating strategy to identify CD45.2+ IL-11R+/+ (control) or IL-11R−/− and CD45.1+ WT control cells among CFSE+ newly recruited cells. (C) Ratio of CD45.2+/CD45.1+ cells among newly recruited CFSE+ cells in WT/IL-11R+/+ and WT/IL-11R−/− mice according to the gating shown in panel (B). Each dot represents one mouse ear. Horizontal bars in red denote the median. Data pooled from two independent experiments. (D) Monocytes (CD11b+Ly6G-) gated with respect to expression of Ly6C and MHC class II in order to identify immature Ly6C+MHCII-, semi-mature Ly6C+MHCII+ and mature Ly6C-MHCII+ monocytes. (E) Ratio of CD45.2+/CD45.1+ cells among newly recruited CFSE+ monocytes in WT/IL-11R+/+ and WT/IL-11R−/−mice within the different cell populations shown in panel (D). Each dot represents one mouse ear. Horizontal bars in red denote the median. Data pooled from two independent experiments. (F) Gating strategy to identify DsRed+ infected cells in WT/IL-11R+/+ and WT/IL-11R−/− mice within the different cell populations shown in panel (B) or (D). (G) Ratio of DsRed+ CD45.2+/CD45.1+ cells among newly recruited CFSE+ monocytes in WT/IL-11R+/+ and WT/IL-11R−/− mice within all CD11b+Ly6G- monocytes according to gating shown in panel (B), and (H) within the different monocyte subsets according to gating shown in panel (D) using controls infected with non-fluorescent L. major wild type parasites. Each dot represents one mouse ear. Horizontal bars in red denote the median. Data pooled from two independent experiments. ns, not significant according to one-way ANOVA with Bonferroni post-test and pairwise comparison. WT, wild type; BM, bone marrow.

To determine whether the IL-11R affects monocyte infection by L. major, we measured the infection rate for the CFSE+ newly recruited monocytes via L. major DsRed fluorescence, and calculated the ratio of infection rates between CD45.1+ and CD45.2+ cells for WT/IL-11R+/+ and WT/IL-11R−/− mice (Fig. 2F). Again, no significant difference was detected, neither for all cells (Fig. 2G), nor for Ly6C+ MHCII-, Ly6C+ MHCII+ or Ly6C- MHCII+ cells when analyzed separately (Fig. 2H). In conclusion, our data argue against a significant role of IL-11R signaling in monocytes during L. major infection.

Discussion

Members of the IL-6 family of cytokines are important regulatory proteins in health and disease, most notably due to their influence on immune cell differentiation, proliferation and apoptosis [1]. Their activity has to be tightly regulated, as overshooting cytokines are associated with many inflammatory diseases, making them suitable therapeutic targets [14]. Consequently, the identification of cytokines that are involved in the pathogenesis of infectious diseases like leishmaniasis could offer novel therapeutic opportunities for patients.

Recently, increased levels of IL-11 mRNA in the lesions of cutaneous leishmaniasis patients infected with L. donovani have been reported [11]. This is of high interest, because important roles for other cytokines during leishmania infection have already been reported. Visceral leishmaniasis represents the most severe form of leishmaniasis and is characterized by high morbidity and finally death of untreated patients. Interestingly, the levels of IL-6, a cytokine closely related to IL-11, correlated with the pathogenesis of the disease as IL-6 levels above 200 pg/ml were strongly associated with death [15]. In a mouse model of cutaneous leishmaniasis, the parasite burden was reduced in IL-6−/− mice compared to wild type animals, although the development of cutaneous lesions did not differ between the mouse groups [16]. In another study, IL-6−/− mice were better able to control the Leishmania infection and displayed earlier and more rapid parasite killing as compared to wild type animals [17].

In contrast to these studies on the role of IL-6, our results indicate that ablation of IL-11R signaling does not influence Leishmania infection in monocytes in vivo. Although monocytes clearly express the IL-11R and are thus target cells of IL-11, our analyses of the different monocyte subsets, namely Ly6C+ MHCII- (recently recruited), Ly6C+ MHCII+ (intermediate) and Ly6C- MHCII+ (matured) monocytes, show no significant differences in the recruitment of these cell populations to the site of infection, nor do they show significant differences in the infection rate of the monocytes when comparing WT/IL-11R+/+ and WT/IL-11R−/− mice. It is tempting to speculate that other Leishmania strains or the analysis of further immunological cell types might reveal an involvement of IL-11 during infection, especially since phagocytic functions are important for efficient L. major entry into host cells [18], [19] and IL-11 has been reported to affect phagocytic functions, e.g. in osteoclasts [20]. However, our data clearly argue against a major role of IL-11 signaling in monocytes during L. major infection.

CRediT authorship contribution statement

Iris Baars: Investigation, Formal analysis, Writing – review & editing, Visualization. Juliane Lokau: Investigation, Writing – review & editing, Visualization. Ina Sauerland: Investigation. Andreas J. Müller: Conceptualization, Writing – review & editing, Supervision, Funding acquisition. Christoph Garbers: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.