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Clinical Infection & Immunity

Original Article

Volume 7, Number 2, September 2022, pages 49-57

Human Vitreous Humor Serves as an Effective Extracellular Matrix for an In Vitro Granuloma System to Study Mycobacterium tuberculosis Infection

Tuberculosis (TB) occurs upon infection with its causative agent, Mycobacterium tuberculosis (Mtb) [1]. The lungs are the primary site of infection by Mtb, and the clinical spectrum ranges from primary TB (5-10% of Mtb infections) to latent asymptomatic infection (LTBI) (95% of Mtb infections) [2, 3]. LTBIs represent a state of equilibrium in which the host can curtail the infection but not eradicate the bacteria. While LTBI has a low risk of mortality, it serves as a reservoir for transmission, and confers risk for TB reactivation stemming from increased host-susceptibility [1, 4]. Approximately 5-15% of latent infections reactivate and manifest as active TB [5].

The host immune response to Mtb infection leads to the formation of hallmark lesions of TB infection, i.e., granulomas, constituted by aggregates of myeloid cells circumscribed by a lymphocytic cuff [6]. The TB granuloma generates an immune microenvironment that prevents extrapulmonary dissemination of Mtb, but provides a niche for its survival, cementing Mtb infection latency [7].

Understanding the host response to Mtb infection is critical to eradicating TB. The development of novel TB therapeutics benefits from a better understanding of the interaction between host-response and Mtb infection. Our current understanding of TB pathophysiology relies on existing in vivo and in vitro models. Various pre-clinical models using various animal species are used extensively, with the limitation in the translatability of findings to the human host [8, 9]. In vitro systems permit dynamic investigations that are not possible clinically or challenging to recreate in animal models. Recommendations for an optimal Mtb-in vitro model include: the use of primary human, mononuclear phagocytic cells and a physiological extracellular matrix (ECM) to mimic host cell survival and Mtb pathogenesis [10]. Current in vitro models for studying granuloma formation, such as cell culture on a collagen matrix, 3D bioelectrospray, organoids, and multicellular lung tissue model, have been insufficient in recreating the immune environment of an infected human host and have model-specific technical challenges [11, 12].

The lung ECM plays a critical role in granuloma formation and Mtb transmission [13]. Commercially available sources of ECM are limited to collagen and synthetic matrices, and although these substances facilitate granuloma formation, they do not accurately reflect the complexity of the human tissue [14]. Recently, the use of vitreous humor (VH) as a potential ECM for cartilage tissue engineering application has been demonstrated [15].

We aimed to develop a novel in vitro model to study Mtb infection in a more physiologically representative microenvironment. Recent applications of VH in tissue engineering highlight its potential to serve as an ECM [15].

In our study, we utilized human vitreous humor (hVH) as a source of ECM to develop an in vitro granulomatous-like system of human monocyte-derived macrophages. Our results demonstrate that hVH can serve as a viable source of ECM to activate macrophages and promote cellular aggregation resembling granuloma formation upon exposure to Mtb.

hVH was harvested from cadavers from the Texas Tech University Health Sciences Center El Paso Willed Body Donor Program. Approval from the Anatomical Board of the State of Texas to use the donor bodies for educational and research purposes is in existence. The eyes were resected using aseptic technique and dissected to extract the vitreous body according to the established protocol by Murali et al [16]. Multiple donor-cadavers were utilized for hVH extraction, with each extraction being isolated. For each experiment (N), only one cadaver-derived hVH was utilized and matched for all samples in that trial.

Human monocytic THP1-dual cells (Invivogen) were differentiated under three conditions upon being plated directly onto hVH. Cells were either untreated, differentiated into macrophages with 10 µM of vitamin D₃ (VD), 1,25-dihydroxy vitamin D (Sigma) or with 5 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 72 h. Cells were then infected with gamma-irradiated Mtb Indo-Oceanic strain (BEI Resources) at a multiplicity of infection (MOI) of 10:1, at OD₆₀₀ 3.13 × 10⁷ micro-organisms, for 3, 7 or 10 days. Uninfected controls for each condition were also followed up for the same period of time to assess any cell aggregates. Cells were observed and photographed daily for the appearance of macrophage aggregates, when number, size and area were measured.

Light microscopy photos of each well were taken daily at × 20 magnification and imported to ImageJ (National Institute of Health). The number of total cellular aggregates was manually counted daily and measurements for area and length on the longest axis were determined using ImageJ’s toolkit. These data were then plotted and analyzed for growth patterns in both aggregate size and area. Metrics were compared between each Mtb condition and control to assess quantified difference. NF-kB and IRF activation were assessed on cell supernatants according to manufacturers’ instructions on a Synergy HTX Multi-Mode plate reader (BioTek). Values were expressed as response ratios compared to the unstimulated wells. Comparisons were performed using a t-test, or a non-parametric test if the data followed or not a normal distribution. Group analysis (of infected vs. Mtb-infected conditions) was performed using ANOVA, for comparison between the different conditions over time.

Before inoculation with Mtb, THP-1 monocyte differentiation towards macrophage cell fate occurred in all conditions, i.e., VH alone, VH + PMA, and VH + VD. Induced differentiation of monocytes to macrophage following a 72-h incubation with PMA and VD was expected. Macrophage differentiation to occur after exposure to VH alone, however, was a remarkable finding (Fig. 1).

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Figure 1. Monocyte differentiation utilizing VH as ECM in vitro. Light microscopy images of day 3 conditions demonstrating characteristic monocyte differentiation into macrophages. Culture-specific differentiated macrophages are highlighted (red circle) with opposing non-differentiated cells to demonstrate change in morphology and signify evidence of monocytic differentiation. ECM: extracellular matrix; VH: vitreous humor.

When the cell culture system was inoculated with Mtb, granuloma-like monocytic aggregation (GLMA) was observed till day 10 (Fig. 2). The total number of GLMAs increased at a variable rate, with a peak observed on day 3 (primary peak). Between days 4 - 5, there is a reduction in the number of GLMAs, followed by an increase on day 6 (secondary peak), and then a consistent decline between days 7 and 10. Uninfected controls, without Mtb, showed no macrophage aggregate formation (n = 5).

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Figure 2. Mtb induces in vitro primary monocytic aggregate cuff formation. (a) In vitro infection-induced monocytic aggregate formation over time upon Mtb inoculation (N = 5) (*NS = not statistically significant). Uninfected samples did not display similar cellular aggregation. (b) Light microscopy imaging with observed THP-1 aggregation in MTb-infected cultures compared to control condition that lacked granuloma-like monocytic aggregation. Mtb: Mycobacterium tuberculosis.

Light microscopy imaging on day 3 demonstrated characteristic macrophage aggregation in Mtb-inoculated conditions (VH, VH + VD, and VH + PMA) compared to control conditions. THP-1 aggregates show a center of less discernible cell composition, possibly containing necrotic cells, which was observed as early as day 3 post-Mtb inoculation in all conditions (Fig. 2).

Comparative analysis of GLMA number, length, and area between Mtb-infected conditions (i.e., VH, VH + VD, and VH + PMA) showed a variable increase of these parameters from days 1 to 3 followed by a primary peak on day 3 (Fig. 3). The highest total number of GLMAs was observed in VH alone condition (51 ± 14.04), followed by VH + PMA (36 ± 7.815), and VH + VD (23 ± 10.54) (Fig. 3a). The largest average length and area were also observed around day 3 (Fig. 3b, c). There was a decrease in these measures between days 4 and 5 followed by a secondary peak observed on day 6. At the secondary peak, VH + VD has the highest number of GLMAs (22.5 ± 7.815), average length (430 ± 152.645 pixels), and area (58,340 ± 21,950.2 pixels²), followed by VH + PMA and VH alone, respectively. From days 7 to 10, we observed a variable decline in all measures and an attenuation of granulomatous response to Mtb infection.

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Figure 3. In vitro GLMA formation upon Mtb inoculation. Average GLMA number (a), average GLMA length (b), and average GLMA area (c) data collected over 10 days in VH, VH + VD, VH + PMA conditions inoculated with Mtb (*N = 5) (*NS = not statistically significant). ANOVA group analysis. GLMA: granuloma-like monocytic aggregate; Mtb: Mycobacterium tuberculosis; VH: vitreous humor; VD: vitamin D3; PMA: phorbol 12-myristate 13-acetate; ANOVA: analysis of variance.

Two main immune response transcription factors, interferon regulatory factor (IRF) and NF-kB pathway activity are needed to mount an inflammatory response against Mtb infection. IRF activation increased from day 0 to day 7 for all conditions with a subsequent decrease by day 10 (Fig. 4a). Although not statistically significant, the VH only condition had the highest IRF activation, compared to VD and PMA (Fig. 4a). VH + VD has the lowest IRF activation.

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Figure 4. Inflammatory transcription factor activation in in vitro GLMA upon Mtb infection. (a) IRF activation measured by luciferase activity (QL), and (b) NF-kB activation measured by alkaline phosphatase (QB). Average response ratios (*N = 3) (*NS = not statistically significant). ANOVA group analysis. GLMA: granuloma-like monocytic aggregate; Mtb: Mycobacterium tuberculosis; IRF: interferon regulatory factor; NF-kB: nuclear factor kappa-B; VH: vitreous humor; PMA: phorbol 12-myristate 13-acetate, VD: vitamin D3; QB: QUANTI-Blue; ANOVA: analysis of variance.

NF-kB pathway activation, on the other hand, was higher on day 3 on VH only condition, followed by a variable decline (Fig. 4b). Notably, the VH + VD and VH + PMA showed a constant NF-kB activation throughout the observed period, despite granuloma formation (Figs. 2 and 4b).

In the lungs, the ECM is a bioactive milieu that plays a critical role in the response to infection and inflammation [17]. The ECM is composed of a myriad of components (predominantly collagen, alongside fibronectin, laminin, proteoglycans, and hyaluronate) that modulate host-pathogen interaction in TB [18]. Cell-matrix interactions affect phagolysosome fusion, pro-inflammatory cytokine secretion, and immune cell activation during Mtb infection [19-21]. Nevertheless, in vitro experiments dissecting intracellular signaling pathways in TB are predominately performed in the absence of an ECM [22]. Current in vitro models that incorporate an ECM to study TB are limited, predominantly using collagen and synthetic matrices [7]. Use of collagen matrices and human peripheral blood mononuclear cells (PBMCs) have shown to develop histological features observed in human TB, such as granulomatous aggregates. However, limitations of these models include low throughput, difficulty in incorporating additional cell types, and lack of natural collagenase to observe ECM destruction, the latter of which is a critical step in Mtb dissemination and transmission [12]. The multicellular lung tissue model and 3D bioelectrospray are modified collagen matrix models that seek to address the limitations of the standard models by incorporating protease-producing cell lineages and increasing translational applications, respectively [12]. Cost-of-use, translation to high throughput, and limited addition of other immune cells remain the most notable challenges [12]. Furthermore, acellular synthetic matrices lack the complexity of human tissue [23].

Current recommendations for an optimal in vitro model prefer longitudinal experiments because the host-pathogen interaction in Mtb infection is often chronic [12]. Our report, however, provides analysis for a relatively short period of time.

Although new small format methods have been proposed to study Mtb infection and granuloma formation, these still rely in the utilization of PBMCs, which may still not traduce into tissue cell types that are involved in real granuloma formation. Also, PBMC from donors will introduce individual variability which could be eliminated using a commercially available cell line. Thus, more-advanced, physiologically comparable in vitro systems are required to study the complex host-pathogen interactions.

The VH is a transparent acellular, avascular ECM situated between the lens and retina, occupying 80% of the eye’s volume [15]. The vitreous gel is highly hydrated (with water constituting 98-99% of the total tissue), and composed of glycosaminoglycans, such as chondroitin sulfate and hyaluronic acid, collagen (predominantly collagen II and IX), and nonstructural proteins [24]. VH possesses immunological components, i.e., immunoglobulins, complement factors, and lysosomal enzymes, with demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria in vitro [25-28]. VH has been previously used as a source for ECM for cartilage tissue engineering application [15]. In our study, we show that hVH can serve as an ECM to produce a cellular aggregate in vitro that mimics the primary Mtb granuloma.

Caseous granulomas are multicellular aggregates that are pathognomonic for TB. The primary cellular components of these aggregate structures are monocyte-differentiated macrophages, which have been shown to be critical in containing Mtb and control mycobacterial proliferation in vivo [6]. It has been shown that human monocytes differentiate into tissue-specific macrophages through interaction with ECM [29]. Our morphological analysis revealed monocyte differentiation into macrophages under all conditions, including VH alone, suggesting that human monocytes interact with hVH and differentiate into macrophages without the need of additional stimuli. An in vitro environment, however, may not fully reflect the complexity of the monocyte-macrophage differentiation process in vivo, which also includes dynamic processes in which monocyte and macrophage-derived molecules contribute to ECM formation, stabilization, and function [30].

VH has been shown to be conducive of granuloma formation in a variety of inflammatory conditions of the eye, such as endophthalmitis, ocular manifestation of sarcoidosis, and Hodgkin’s lymphoma [31-33]. We report the formation of granulomatous aggregates in the highest numbers on macrophage cell cultures inoculated with Mtb in VH alone, VH with PMA, and VH with VD, respectively on day 3. A decrease in GLMA number was observed with time progression, with a secondary peak of GLMA formation on day 7 with VH alone and VH + PMA, and as early as day 6 with VH along with VD. VD has been linked to play a role in macrophage activation and granuloma formation [34]. Recent evidence suggests that Mtb orchestrates the early events of granuloma formation for its survival [35], freely proliferating inside of granulomas and utilizing virulence factors to promote granuloma dissociation [35]. As TB progresses, necrotic cell death results in bacterium leakage into the growth permissive extracellular environment and prevents Mtb from being phagocytosed by new macrophages, resulting in a decrease in the number of cell aggregates [6]. Further research is needed to assess these phenomena.

The molecular mechanism of granuloma formation is driven by macrophage-derived transcription factors including NF-kB and IRFs [36]. Activation of NF-kB and IRF pathways upregulate expression and secretion of pro-inflammatory cytokines (such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, etc.) which interact with the ECM driving the immune response to Mtb [36]. Notably, NF-kB mediates bacterial load within a granuloma and granuloma size [37], and NF-kB mutants disrupt mycobacterial immunopathology in vivo resulting in disseminated TB sequelae. We observed NF-kB and IRF activation in VH-differentiated macrophages exposed to Mtb, with decreasing levels of NF-kB activation corresponding to lower number of and lesser area of GLMA. Inhibition of NF-kB decreases survival of intracellular Mtb in human macrophages, thus providing an avenue for future research to determine relationship between VH, ECM, NF-kB activation and Mtb survivability [38].

VH-differentiated macrophages also showed activation of IRF in response to Mtb. Current data suggest that IRFs play an important role in regulation macrophage polarization, granuloma necrosis, antigen presentation in myeloid cells during tuberculosis, and apoptosis of ECM in human mammary epithelial cells [39-42]. The formation of macrophage aggregates with denuded centers on day 3 provide may be associated with IRF activation, although the specifics of this interaction will need future investigation. Further study should broaden pathogen analysis with non-pathogenic Mtb species to further increase validity of findings. Moreover, we acknowledge that use of VH is susceptible to the patient-specific variability, much like the utilization of PBMCs. However, recent literature has suggested protocols to construct artificial VH which curtails this drawback and provides robust exploration through a commercially available product [43].

In conclusion, our study demonstrates that hVH serves as an effective ECM to study macrophage activation and granuloma-like macrophage aggregates upon Mtb infection in vitro. Future projects will seek to address translatability alongside pharmacotherapy, and molecular analysis.

Acknowledgments

Special recognition to donors participating in the Texas Tech University Health Sciences Center El Paso Willed Body Program, for allowing us to pursue scientific projects from cadaveric tissues.

Financial Disclosure

No external funding.

Conflict of Interest

The authors declare no conflict of interest.

Informed Consent

Not applicable.

Author Contributions

SB, JR, and JB performed the experiments. SB and JR acquired data. JA and RB performed eyes and VH extraction. SB, JR, and JC analyzed the data. JC interpretation of data. SB, JR, and JC wrote the manuscript.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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