Anti-tumor necrosis factor alpha reduces the proangiogenic effects of activated macrophages derived from patients with age-related macular degeneration

Patients

Two elderly (92 and 86 years of age) male patients with nvAMD were recruited from the Retina Clinic in the Department of Ophthalmology at Hadassah-Hebrew University Medical Center. The patients’ diagnosis of AMD was based on criteria established by the Age-Related Eye Disease Study (AREDS) [23], and CNV was confirmed using fluorescein angiography and optical coherence tomography. Overall, the lesions comprised at least 50% active CNV and <25% subretinal hemorrhage. The patients did not present with any other retinal disease or any other possible causes of CNV, such as myopia, trauma, or uveitis. In addition, the patients did not present with any major systemic illness, such as cancer, autoimmune disease, congestive heart failure, or unmanaged diabetes.

Ethics

All experimental protocols and studies involving human subjects were approved by the local Committee on Research Involving Human Subjects of the Hebrew University-Hadassah Medical School, the Helsinki Committee of Hadassah Medical Organization, and the Israel Ministry of Health’s Helsinki Committee for Genetic Experiments on Human Subjects (File #22–03.08.07). Patients and control subjects provided written informed consent in accordance with the tenets of the Declaration of Helsinki prior to participating in the study, in accordance to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO). All methods used in the study were performed in accordance with approved study guidelines while ensuring the participants’ privacy.

Preparation of macrophages

Blood samples (30 ml each) from the patients with nvAMD were collected into EDTA-containing tubes (BD Biosciences). Monocytes were isolated from the whole blood samples, differentiated into M0 macrophages, and then activated to form M1 macrophages by stimulation with IFN-γ and LPS as previously described [11,24-28]. We chose this specific macrophage subtype because we previously found that M1 macrophages cause increased neovascularization in vitro and in vivo [11]. The medium was harvested from the cultured macrophages and stored at −20 °C for use in the choroid sprouting assay (CSA). Macrophages were collected using 0.25% trypsin (Sigma-Aldrich, Munich, Germany), washed with RPMI 1640 medium (Biological Industries Israel Beit Haemek Ltd) containing fetal calf serum (FCS), washed 3 times with phosphate-buffered saline (1X PBS; 135 mM NaCl, 2.7 mM KCl, 10 mM Na₂PO₄, 1.5 mM KH₂PO₄, pH 7.4), and used immediately in the in vivo experiments.

Animal experiments

All protocols involving animals were approved by the Authority for Biologic and Pre-Clinical Models (ABBM) and the University Ethics Committee for the Care and Use of Laboratory Animals of Hebrew University, which is certified by the Association for Accreditation of Laboratory Animal Care (AAALAC; approval number: MD-16–14796–3, NIH approval number: OPRR-A01–5011). All researchers working with laboratory animals were approved by the ethics committee of the ABBM. All guidelines with respect to the humane and ethical treatment of laboratory animals based on the Association for Research in Vision and Ophthalmology (ARVO) were strictly followed, and all methods used in this study were performed in accordance with approved study guidelines.

Choroid sprouting assay

This ex vivo angiogenesis assay was performed as previously described [29] to assess the putative angiogenic effects of candidate cytokines collected from the supernatant of cultured M1 macrophages prepared from patients with nvAMD as described above [11]. Cytokines were added to the medium at the maximum concentration based on previous enzyme-linked immunosorbent assay (ELISA) results measured using the culture medium of M1 macrophages [11]. Specifically, the following five cytokines (all from PeproTech, Rocky Hill, NJ) were tested at the indicated concentrations (n = 8 replicates for each cytokine): VEGF, 0.25 ng/µl; IL-8, 0.50 ng/µl; IL-6, 2.50 ng/µl; IL-1β, 0.05 ng/µl; and TNF-α, 0.85 ng/µl. Where indicated, anti-TNF-α (infliximab, 100 µg/µl; Remsima, Celltrion Inc., Incheon, South Korea) and anti-VEGF (aflibercept, 40 µg/µl; Bayer Pharma AG, Berlin, Germany) were included in the presence or absence of M1 macrophage‒derived medium and were used at concentrations based on previous studies [30].

For the CSA assay, C57BL/6J mice (4-6 weeks old) were euthanized by cervical dislocation following anesthesia with inhalation liquid Isoflurane USP 100% (provided by Piramal pharma solutions, Mumbai, India), and the eyes were removed immediately and placed in ice-cold endothelial cell growth medium (ECGM, Cat. C-22010; PromoCell, Heidelberg, Germany) containing 100 units/ml penicillin-streptomycin (Biological industries, Beit Haemek, Israel) and 1% glutamine (Biological industries). The choroid-sclera complex was then gently dissected along with the retinal pigment epithelium (RPE) layer. The tissue was then cut into 5-6 1-mm long pieces, which were embedded in 30 µl of growth factor‒reduced Matrigel (Cat. 354230, BD Biosciences, Cowley, Oxford, UK354230) in 24-well plates. The thickness of the Matrigel layer was approximately 0.4 mm. The plates were then incubated without medium for 10 min at 37 °C in 5% CO₂ to solidify the Matrigel, after which 500 µl ECGM containing 2.5% supplement mix (C-9215, PromoCell), 5% FCS, 100 units/ml penicillin-streptomycin, and 1% glutamine was added to each well. For the experimental groups containing the supernatant from cultured macrophages, the medium was replaced with 250 µl of this supernatant. The medium in each well was changed every 3 days, and the cultures were fixed with 4% paraformaldehyde (PFA) on day 8. The cultures were viewed using an inverted Olympus CKX41 microscope, and images were captured using a DP70 digital camera (Olympus, Tokyo, Japan).

ImageJ (NIH, Bethesda, MD) was used to quantify the sprouting area [31]. The sprouting area was selected using the software and measured after the choroid tissue was excluded. The background (a control well in each plate containing medium only) was subtracted from each sample. For each well, the ratio between the experimental group and its respective control sample from the same eye was calculated, and replicates were averaged. Although we did not label the sprouting cells in the CSA, previous works have suggested that these sprouts are mostly composed of endothelial cells [2-4].

Laser-induced CNV

For the LI-CNV model, Long-Evans rats (8–12 weeks old) were anesthetized with an intraperitoneal injection of a mixture containing 85% ketamine (Bedford Laboratories, Bedford, OH) and 15% xylazine (InoVet, Arendonk, Belgium). Topical anesthesia (Oxybuprocaine HCL 0.4%; Fisher Pharmaceuticals, Tel-Aviv, Israel) was applied to each eye 10 min before intravitreal injection and laser photocoagulation.

Laser photocoagulation burns (five to seven spots per eye) were applied as previously described [32]. Two days later, the respective compounds were administered by intravitreal injection using a PLI-100 Pico-Injector (Medical System Corp., Greenvale, NY) to induce CNV [33]; these injections were repeated every 2 days for a total of 10 days. Where applicable, macrophages were injected only once (2 days after laser photocoagulation). The entire procedure, including evidence showing lack of a xenograft-induced immune response, has been published previously [11].

The following control group and five experimental groups were included in this study: PBS (4 µl, control); infliximab (1 µl of a 100 µg/µl solution); aflibercept (1 µl of a 100 µg/µl solution); infliximab plus 10⁵ M1 macrophages (1 µl and 4 µl, respectively); aflibercept plus 10⁵ M1 macrophages (1 µl and 4 µl, respectively); and 10⁵ M1 macrophages (4 µl). The concentration of each compound injected was based on previous studies [34]. For each group, both eyes in four rats were injected (for a total of eight eyes per group). After each injection, antibiotic ointment (5% chloramphenicol) was applied to each eye. After the final injections (i.e., 10 days after laser photocoagulation), choroid-RPE and retinal flat mounts were prepared as previously described [11,35] and analyzed as described below.

Quantification of CNV

Choroid-RPE flat mounts were fixed for 1 h in 4% PFA and suspended overnight in Isolectin GS-IB4 Alexa Fluor 594 staining solution (Molecular Probes, Eugene, OR) containing 200 mM NaN₃ and 1 mM CaCl₂. The flat mounts were then washed six times for 20 min each in PBS and mounted on a slide using mounting medium. The area of CNV surrounding each laser injury was measured using ImageJ as previously described [11].

Basic statistical analyses

Data were analyzed using the biostatistics software package InStat (GraphPad, San Diego, CA). Differences with a p-value <0.05 were considered statistically significant. Outliers (±2 SD) were excluded from the statistical analysis (less than 5%) [39]. Appropriate statistical tests were used based on the results of a test for normality, the sample distribution, and parameters.

Analysis of macrophage modulation

Using data from the present study and a previous study [11], we examined the role of age, gender, disease status (nvAMD versus control), and macrophage subtype (M0 versus M1 versus M2) on mRNA levels, protein levels, and proangiogenic properties in the CSA and LI-CNV models. We included in the analysis the protein levels of PDGF, TNF-α, VEGF, MCP1 (i.e., CCL2), and ICAM (measured using ELISA), as well as the mRNA levels of PDGF (Gene ID 5154, OMIM 173430), TNF-α (Gene ID 7124, OMIM 191160), and VEGF (Gene ID 3383, OMIM 147840) measured using qPCR) of all patient-derived polarized human macrophages. These data were obtained from our previous study [11] and included 34 patients with nvAMD (21 women and 13 men) with a mean (± standard error of the mean [SEM]) age of 75.9±1.50 years (range: 59–93 years) and 25 age-matched unaffected controls (ten women and 15 men) with a mean age of 72.3±1.70 years (range: 59–89 years).

We then evaluated the contribution of the patient (“Cell Origin”) and the laboratory manipulation of cultured macrophages (“Environment”) to the protein and mRNA levels measured in the macrophages. These contributions were compared using an analysis of variance (ANOVA) with the software program R (and R studio) together with the “reshape2” package. We then calculated the percentage of variance that was due to age, gender, disease status, and environment (and the interaction between cell origin and environment), as well as any unexplained variance, using a mixed-design ANOVA with repeated measures in R.

TNF-α and anti-TNF-α therapy increases and decreases, respectively, the proangiogenic effects of human M1 macrophages on choroid sprouting in an ex vivo model

First, using an ex vivo choroid sprouting assay, we examined whether the addition of exogenous cytokines affects neovascularization. We found that addition of TNF-α to the culture medium statistically significantly increased sprouting compared to the untreated wells (Figure 1A). Specifically, the mean (± SEM) ratio of the relative sprouting area between the TNF-α-treated and untreated wells was 1.6±0.2 (n = 8; p=0.01, Student t test). In contrast, addition of either IL-6 or IL-8 statistically significantly decreased the sprouting area (Figure 1A), with a relative ratio between treated and untreated wells of 0.64±0.1 (n = 9; p=0.01, Student t test) and 0.47±0.14 (n = 8; p=0.007, Student t test), respectively. Addition of either IL-1β or VEGF had no effect on choroid sprouting (Figure 1A). The lack of an effect of VEGF in this model may have been due to the high levels of VEGF present in the growth medium [29], which may have obscured any possible effect of VEGF added to the medium.

Next, we examined whether supernatant collected from cultured M1 macrophages affects choroid sprouting and found that addition of this supernatant statistically significantly increased the sprouting area to 1.33±0.12 (n = 7, p=0.04, Student t test) relative to the untreated wells (Figure 1B). Moreover, this increase was prevented by adding either anti-TNF-α or anti-VEGF to the M1 supernatant, although treatment with either anti-TNF-α or anti-VEGF alone had no statistically significant effect on choroid sprouting (Figure 1B).

Taken together, these findings suggest that TNF-α—and possibly VEGF as well—mediates the proangiogenic effect of M1 macrophages in choroid sprouting. Moreover, the increase in sprouting induced by M1 supernatant, but not VEGF alone, suggests that macrophages can induce choroid sprouting via a VEGF-independent pathway.

Anti-TNF-α therapy prevents the proangiogenic effect of human MI/L macrophages in an in vivo model

Next, we used an in vivo model of LI-CNV to examine whether intravitreal injections of anti-TNF-α can reduce the proangiogenic effects of M1 macrophages. We found that intravitreal adoptive transfer of M1 macrophages statistically significantly increased the CNV area compared to controls (Figure 2). Moreover, this increase was eliminated by coinjecting either anti-TNF-α or anti-VEGF together with M1 macrophages. Consistent with the ex vivo results, we also found that injecting anti-TNF-α alone had no effect on CNV (Figure 2). Interestingly, and in contrast with the ex vivo results, we also found that intravitreal injection of anti-VEGF alone statistically significantly reduced the CNV area (Figure 2).

Cell origin and environment can explain part of the variance in TNF-α expression at the mRNA and protein levels

Next, we examined our previous data regarding the expression profile of activated macrophages obtained from patients and controls [11] to determine whether cell origin, or the environment, or both can explain the variance in the expression of TNF-α and other cytokines in macrophages, thus indicating that expression is modifiable. At the mRNA level (Figure 3A), we found that 29% of the variance in the expression of TNF-α, VEGF, and PDGF measured in three macrophage subtypes cultured from patients with nvAMD (n = 7) and age-matched controls (n = 9) was explained by cell origin (i.e., patient-derived factors). However, 16%, 20%, and 9% of the variance in the TNF-α, VEGF, and PDGF mRNA levels, respectively, was explained by environmental factors (i.e., the cell culture conditions used to generate the macrophage phenotypes). Finally, 55%, 51%, and 62% of the variance in the TNF-α, VEGF, and PDGF mRNA levels, respectively, was either unexplained or was explained by the interaction between the cell origin and the environment.

Using a mixed-design ANOVA with repeated measures, we combined non-modifiable factors, such as the patient’s age, gender, and disease status, and found that the interaction between the environment and the cell origin and the environment alone explained 58%, 65%, and 21% of the variance in the TNF-α, VEGF, and PDGF mRNA levels, respectively. Finally, 13%, 6%, and 50% of the variance in the TNF-α, VEGF, and PDGF mRNA levels, respectively, was unexplained (Figure 3A). These results suggest that more than 50% of the variance in the expression of TNF-α and VEGF at the mRNA level can be modified by factors such as cell origin and environment.

With respect to the variance in protein levels (based on 13 patients with nvAMD and 13 age-matched controls), we found that only 19% of the variance in the TNF-α protein was explained by cell origin, whereas 59% of the variance was explained by the environment (Figure 3B). In contrast, 58%, 95%, 56%, and 61% of the variance in VEGF, PDGF, ICAM, and MCP1, respectively, was explained by cell origin (Figure 3B). These results suggest that among these five cytokines, the expression of TNF-α is the most modifiable in activated macrophages.

Macrophage function in two models of CNV is modifiable

Using a mixed-design ANOVA with repeated measures, we found that macrophage function was modifiable in the in vivo and ex vivo models of CNV. Specifically, we found that cell origin explained only 17% and 35% of the variance in the effect of the three macrophage subtypes in the LI-CNV and CSA models, respectively (eight patients with nvAMD and nine age-matched controls), whereas 55% and 50%, respectively, was explained by environment and the interaction between the cell origin and the environment (Figure 4).