A Heterotetramer of Galectin-1 and Galectin-3 Mitigates Localized Sterile Inflammation

Introduction:: Chronic inflammation is associated with the onset and progression of numerous diseases, such that it is considered the leading cause of disability and mortality worldwide [1]. Current treatment regimens for chronic inflammation involve long-term systemic use of conventional anti-inflammatory drugs, such as glucocorticoids, that are associated with a multitude of adverse health effects. Furthermore, chronic inflammation often manifests locally. Biotherapeutics capable of mitigating inflammation locally are attractive alternatives to conventional drugs. Galectin-1 (Gal1) and Galectin-3 (Gal3) are natural immunomodulatory proteins that are attractive candidates to resolve localized chronic inflammation by mimicking natural mechanisms of immune regulation. However, the unstable native quaternary structures of Gal1 and Gal3 hinder their immunotherapeutic potential. To address this instability, we created a stable synthetic heterotetramer of Gal1 and Gal3 using an α-helical coiled-coil scaffold [2]. Known as “G1/G3 Zipper”, this construct is more potent than either of the GalX proteins in vitro [2]. Here, we report that G1/G3 Zipper effectively mitigates localized sterile inflammation in two well-characterized inflammation models: λ-Carrageenan and imiquimod-induced psoriasis.

Materials and Methods:: G1/G3 Zipper (Fig. 1A) was expressed and purified from E. coli, as reported [2]. G1/G3 Zipper activity was confirmed using an in vitro Jurkat T cell agglutination and apoptosis assay, as reported [3]. For the sterile inflammation model, C57BL/6J mice received an injection of 1% λ-Carrageenan in one back foot at t = 0. At t = 24 h, the control group received an injection of PBS, while the experimental group received an injection of 0.3 µg/µL G1/G3 Zipper (Fig. 1B). Paw diameters were measured using digital calipers at t = 0, 6, 24, 48, 72, and 96 hours (Fig. 1C). At t = 96 h, tissue was collected, processed, and stained with H&E by the UF Pathology core, and scored for thickness and immune cell infiltrate as reported [4].

For the psoriasis model, an electric razor and depilatory cream were used to remove a patch of hair on the backs of C57BL/6J mice. Mice then received daily topical doses (62.5 mg) of a commercially available imiquimod cream (5%) for 6 consecutive days. Mice were injected subcutaneously on day 4 with five spatially distributed injections (50 µL/injection) of either PBS or 21 µM G1/G3 Zipper (Fig. 1B). Clinical skin scores were evaluated through day 7 using a modified psoriasis area and severity index (PASI) based on the degrees of epidermal erythema, induration, and scaling (Fig. 1D).


Results, Conclusions, and Discussions:: Results & Discussion: G1/G3 Zipper suppressed paw inflammation when injected subcutaneously at the challenge site during the early edema phase (t = 24 h) that follows λ-Carrageenan injection (Fig. 1G). Iqbal et al. demonstrated an oxidation resistant variant of Gal1, now the gold-standard, suppressed edema but not inflammation in the λ-Carrageenan model [5]. In stark contrast, G1/G3 Zipper suppressed this secondary inflammation phase, demonstrating greater efficacy in vivo than the current standard Gal1 variant. Histological analysis demonstrated a significant reduction in immune cell infiltrate in G1/G3 Zipper treated tissue as compared to the vehicle [Fig. 1E,F].

In the imiquimod-induced psoriasis model, G1/G3 Zipper significantly decreased the PASI score within 48 h, approaching near 50% of baseline by day 7 when compared to the vehicle alone (Fig. 1H). Shi et al. demonstrated that intracutaneous injection of recombinant Gal3 (rGal3) could decrease inflammation in imiquimod-induced psoriasis in Gal3-/- mice; however, no significant improvement was observed in wild-type mice [6]. Thus, G1/G3 Zipper shows potential as a better biotherapeutic to treat psoriasis based on PASI scoring than WT-Gal3.


Neutrophils are a shared cell mediator of inflammation following λ-Carrageenan injection and psoriasis induced by imiquimod treatment. Gal1 has been shown to signal neutrophil phosphatidylserine (PS) exposure, which flags these cells for clearance via efferocytosis during resolution of inflammation. G1/G3 Zipper induced PS exposure more effectively than wild-type Gal1 or a stable Gal1 homodimer (data not shown). When injected with rGal3, Gal3-/- mice have also shown a reduction in neutrophil infiltration in vivo [6].

Conclusions: Our in vivo data demonstrate G1/G3 Zipper effectively mitigates inflammation in both the λ-Carrageenan and imiquimod-induced psoriasis models. Our conclusions are further supported by our in vitro work (data not shown) demonstrating G1/G3 Zipper can induce neutrophil phosphatidylserine exposure, a mechanism for neutrophil clearance that is an important aspect in resolving inflammation.

Impact: Creating synthetic galectin constructs with stable quaternary structures can lead to potent new anti-inflammatory biotherapeutics.


Acknowledgements (Optional): :

References (Optional): : [1] Furman, D., Campisi, J., Verdin, E., et al. Nat. Med. 25, 1822-1832 (2019)

[2] Fettis, M.M., Farhadi, S.A., Hudalla, G.A. Biomater. Sci. 7, 1852–1862 (2019)

[3] Farhadi, S.A., Fettis, M.M., Liu, R., et al. Front. Chem. 7, 1-9 (2020)

[4] Morales, B.S., Liu, R., Olguin, J., et al. Biomater. Sci. 9, 2494–2507 (2021)

[5] Iqbal, A.J. et al. J. Immunol. 191, 171–177 (2013).

[6] Shi, Z.R., et al. J. Autoimmun. 89, 30–40 (2018)