Targeting CARD9 with Small-Molecule Therapeutics Inhibits Innate Immune Signaling and Inflammatory Response to Pneumocystis carinii β-Glucans

Pneumocystis jirovecii, the opportunistic fungus that causes Pneumocystis pneumonia (PCP) in humans, is a significant contributor to morbidity and mortality in immunocompromised patients. Given the profound deleterious inflammatory effects of the major β-glucan cell wall carbohydrate constituents of Pneumocystis through Dectin-1 engagement and downstream caspase recruitment domain-containing protein 9 (CARD9) immune activation, we sought to determine whether the pharmacodynamic activity of the known CARD9 inhibitor BRD5529 might have a therapeutic effect on macrophage innate immune signaling and subsequent downstream anti-inflammatory activity.

ABSTRACT

Pneumocystis jirovecii, the opportunistic fungus that causes Pneumocystis pneumonia (PCP) in humans, is a significant contributor to morbidity and mortality in immunocompromised patients. Given the profound deleterious inflammatory effects of the major β-glucan cell wall carbohydrate constituents of Pneumocystis through Dectin-1 engagement and downstream caspase recruitment domain-containing protein 9 (CARD9) immune activation, we sought to determine whether the pharmacodynamic activity of the known CARD9 inhibitor BRD5529 might have a therapeutic effect on macrophage innate immune signaling and subsequent downstream anti-inflammatory activity. The small-molecule inhibitor BRD5529 was able to significantly reduce both phospho-p38 and phospho-pERK1 signaling and tumor necrosis factor alpha (TNF-α) release during stimulation of macrophages with Pneumocystis cell wall β-glucans.

TEXT

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule that is critical for key signaling pathways initiated through C-type lectin receptors (CLRs) that participate in host responses during many fungal infections (1–6). BRD5529, a small-molecule inhibitor shown previously to selectively bind CARD9 and inhibit TRIM62 binding (7), can lead to decreased activity in host inflammatory responses. Accordingly, this agent was employed to determine whether it might serve as an alternate agent to alleviate the robust proinflammatory effects induced by Pneumocystis β-glucans through Dectin-1/CARD9 innate cell signaling. Indeed, appropriately timed therapeutic inhibition of CARD9 signaling might help mitigate subsequent proinflammatory events caused by anti-Pneumocystis pneumonia (PCP) therapy, which results in the killing of organisms and cell wall exposure and of exposed, highly inflammatory β-glucan molecules (8–14).

Briefly, 2 × 10⁵ RAW 264.7 macrophages (American Type Culture Collection) were plated per well in duplicate in 96-well microtiter plates and incubated for 4 h. Next, BRD5529, purchased from Sigma-Aldrich, was preincubated with the RAW 264.7 cells for 60 min. Subsequently, 100 μg/ml of Pneumocystis carinii β-glucans (15) was added to the wells, and the plates were centrifuged at 500 × g to synchronize macrophage-carbohydrate interactions. Plates were then incubated at 37°C for 60 min. Subsequently, the cells were washed with 1× phosphate-buffered saline (PBS) and lysed, and protein quantification was performed. Equal quantities of proteins were loaded and separated by polyacrylamide gel electrophoresis (PAGE). Separated proteins were transferred to nylon for Western blotting and incubated with antibodies to phospho-ERK1/2 (extracellular signal-regulated kinase 1/2) or phospho-p38 as well as total ERK1/2 and p38 (Santa Cruz Biotechnology) to demonstrate equal protein loading. Quantification of differences in protein phosphorylation was conducted using Image Studio Lite (LI-COR). All experiments were repeated 5 to 6 times. MAPK (mitogen-activated protein kinase) activation is involved in the macrophage response to Pneumocystis infection (16). The CARD9 adaptor protein, through its interaction with the BCL10-MALT1 complex, is implicated in MAPK activation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors in myeloid cells (17). We initially observed that the activation of MAPK indeed occurs in macrophages in the presence of Pneumocystis organisms and that this activity is impaired in the absence of CARD9 (18).

Here, we demonstrate that BRD5529 can indeed significantly inhibit P. carinii β-glucan-induced downstream CARD9 phosphorylation of both p38 and pERK1/2 in a dose-dependent manner and also with BRD5529 concentrations similar to the ones previously reported for primary bone marrow-derived dendritic cells (BMDCs) stimulated with scleroglucan (Fig. 1 and 2) (7). We did not observe any evidence of cell toxicity at the concentrations of BRD5529 used in these studies. Next, we determined whether BRD5529 inhibition of CARD9 would not only result in decreased MAPK phosphorylation but also lead to reduced secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Indeed, we previously demonstrated that this cytokine is markedly increased in the presence of Pneumocystis organisms or the organisms’ β-glucan cell wall components (13, 19–22). As similarly noted above, the absence of CARD9 in a mouse model of PCP resulted in a profound decrease in this vital proinflammatory cytokine (18). To determine this, we plated RAW 264.7 macrophages as described above. After 4 h, BRD5529 was incubated with the RAW 264.7 cells for 60 min. The supernatant was removed, and next, 100 μg/ml of P. carinii β-glucans plus the BRD5529 compound were added to the cells, and the cells were incubated at 37°C for 18 h. The supernatants were then collected and assayed for TNF-α by an enzyme-linked immunosorbent assay (ELISA) (13). As demonstrated in Fig. 3, BRD5529 significantly reduced TNF-α release in a dose-dependent fashion. We and others have shown the importance of CLRs in host immune recognition in PCP. These include Dectin-1, Dectin-2, and Mincle (12, 19, 20, 23–25). Recently, we reported that the CLRs Dectin-2, Mincle, and macrophage C-type lectin (MCL) can all bind P. carinii homogenates (26). Based on these results, this fungal organism, and its cell wall constituents, can be recognized by the host via a variety of CLRs that all signal through the CARD9 axis to initiate host inflammatory cascades. Indeed, for example, we and others have demonstrated the importance of P. carinii β-glucan via Dectin-1/CARD9 activation (8, 10, 12, 19, 20, 23).

FIG 1.

(A) BRD5529 can significantly inhibit P. carinii (P.c.) β-glucan-induced ERK1/2 phosphorylation in RAW 264.7 cells in a dose-dependent manner. Shown is a blot representative of results from 5 separate experiments. (B) The phospho-ERK1/2 signals were quantified with Image Studio Lite software and normalized to total ERK1/2 levels. For multigroup data, initial analysis was first performed by analysis of variance (ANOVA) to determine overall differences. If ANOVA indicated overall differences, subsequent subgroup analysis was then performed by Newman-Keuls multiple-comparison testing. Statistical analysis was performed using Prism version 8.4.3 software (GraphPad Inc.). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2.

(A) BRD5529 can significantly inhibit P. carinii β-glucan-induced p38 phosphorylation in RAW 264.7 cells in a dose-dependent manner. Shown is a blot representative of results from 6 separate experiments. (B) The phospho-p38 signals were quantified with Image Studio Lite software and normalized to total p38 levels. For multigroup data, initial analysis was first performed by ANOVA to determine overall differences. If ANOVA indicated overall differences, subsequent subgroup analysis was then performed by multiple-comparison testing. **, P < 0.01; ***, P < 0.001.

FIG 3.

BRD5529 significantly dampens RAW 264.7 cell production of TNF-α in vitro in the presence of P. carinii β-glucans. Data are the means ± standard errors of the means (SEM) for at least 7 separate experiments. For multigroup data, initial analysis was first performed by ANOVA to determine overall differences, followed by multiple-comparison testing. *, P < 0.05; ***, P < 0.001; ns, not significantly different.

Taken together, these data suggest promising preliminary evidence that therapeutically targeting the CARD9 pathway in those immunocompromised patients with PCP undergoing antifungal treatment may provide an additional beneficial reduction of the deleterious robust inflammatory responses associated with Pneumocystis pneumonia.