What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Wnt Signaling from Macrophages: Mechanisms and Roles

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Wnt Signaling from Macrophages

What is Wnt Signaling?

Wnt signaling is a highly conserved cell-to-cell communication system that controls gene expression, cell polarity, proliferation, and differentiation across metazoans. The name derives from the fusion of Wingless (a Drosophila segment polarity gene) and Int-1 (a mouse mammary oncogene). Wnt ligands are secreted glycoproteins, typically 350–400 amino acids in length, that bind to Frizzled (FZD) receptors on the plasma membrane of recipient cells. This binding initiates intracellular cascades that converge on transcriptional regulators such as β-catenin (canonical pathway) or on cytoskeletal and calcium-dependent effectors (non-canonical pathways). The pathway is reviewed in depth in the Wnt Signaling Pathway reference.

Wnt signaling is not a single linear cascade but a network of overlapping branches. The canonical pathway stabilizes β-catenin, allowing it to enter the nucleus and activate T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. Non-canonical pathways operate independently of β-catenin and include the planar cell polarity (PCP) pathway and the Wnt/calcium pathway. The choice of pathway depends on the Wnt ligand identity, the receptor context (FZD paralogs, co-receptors such as LRP5/6 or ROR1/2), and the cellular environment.

Macrophages as Wnt-Producing Cells

Macrophages are innate immune cells best known for phagocytosis, antigen presentation, and cytokine secretion. However, they also function as significant sources of Wnt ligands in both steady-state and diseased tissues. Tissue-resident macrophages—such as Kupffer cells in the liver, microglia in the brain, and osteoclast precursors in bone—secrete Wnts that act on neighboring epithelial, stromal, and stem cells. This paracrine activity is essential for maintaining tissue architecture, supporting stem cell niches, and coordinating repair responses after injury.

The importance of macrophage-derived Wnts is most evident in contexts where macrophage depletion leads to tissue dysfunction. For example, in the intestinal crypt, macrophages adjacent to the stem cell zone secrete Wnt ligands that help sustain Lgr5+ stem cells. In the liver, Kupffer cell–derived Wnts support hepatocyte proliferation during regeneration. In tumors, tumor-associated macrophages (TAMs) secrete Wnts that promote cancer cell stemness and chemoresistance. Understanding the molecular mechanisms of Wnt production and secretion from macrophages is therefore central to both developmental biology and translational medicine.

Molecular Mechanisms of Wnt Secretion and Signaling

Wnt Ligand Processing and Secretion

Wnt proteins undergo a series of post-translational modifications that are essential for their secretion and function. The process begins in the endoplasmic reticulum (ER) where Wnts are lipid-modified by the acyltransferase Porcupine (PORCN). PORCN attaches a palmitoleate group to a conserved serine residue (e.g., Ser209 in human WNT3A). This lipid modification is required for Wnt binding to its receptor Frizzled and for its secretion. Without PORCN activity, Wnts remain trapped in the ER and are rapidly degraded.

After lipid modification, Wnts bind to Wntless (WLS, also called GPR177 or Evi), a dedicated cargo receptor that transports them from the Golgi to the plasma membrane. At the cell surface, Wnts are released into the extracellular space, where they can diffuse locally or be packaged into exosomes. The secretion process is tightly regulated; WLS is recycled back to the Golgi via the retromer complex, and disruption of retromer function leads to Wnt secretion defects.

In macrophages, the secretion machinery is identical to that in other Wnt-producing cells, but the regulation differs. Macrophages express multiple WNT genes, including WNT2, WNT3A, WNT5A, WNT7B, and WNT10A, depending on tissue context and activation state. For example, pro-inflammatory macrophages (M1) tend to upregulate WNT5A, whereas anti-inflammatory macrophages (M2) often express WNT2 and WNT7B. The lipid-modified Wnts are released into the extracellular milieu where they can act on adjacent cells in a paracrine manner.

Canonical β-Catenin Pathway

The canonical Wnt pathway is activated when a Wnt ligand binds to a Frizzled receptor and the co-receptor LRP5/6 (low-density lipoprotein receptor-related protein 5/6). This binding triggers phosphorylation of LRP6 by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), creating a docking site for Axin. The recruitment of Axin to the receptor complex disrupts the β-catenin destruction complex, which normally consists of Axin, adenomatous polyposis coli (APC), GSK3, and CK1.

In the absence of Wnt, the destruction complex phosphorylates β-catenin, marking it for ubiquitination by β-TrCP and subsequent proteasomal degradation. When Wnt signaling is active, β-catenin is no longer phosphorylated and accumulates in the cytoplasm. Stabilized β-catenin translocates to the nucleus, where it displaces co-repressors from TCF/LEF transcription factors and recruits co-activators such as CBP/p300. This leads to transcriptional activation of target genes including MYC, CCND1 (cyclin D1), and AXIN2.

Macrophage-derived Wnts that activate the canonical pathway include WNT1, WNT2, WNT3A, and WNT7B. These ligands are particularly important in stem cell maintenance and tissue regeneration. For instance, in the intestinal crypt, macrophage-secreted WNT2B supports the proliferative capacity of Lgr5+ stem cells. In the liver, Kupffer cell–derived WNT3A promotes hepatocyte proliferation after partial hepatectomy.

Non-Canonical Pathways (PCP and Ca2+)

Non-canonical Wnt signaling does not involve β-catenin stabilization. The planar cell polarity (PCP) pathway is activated by Wnt ligands such as WNT5A and WNT11 binding to Frizzled receptors and co-receptors ROR1/ROR2. This activates the small GTPases RhoA and Rac1, leading to activation of Rho-associated kinase (ROCK) and JNK. The result is cytoskeletal reorganization, cell polarity, and changes in cell migration. In macrophages, autocrine WNT5A signaling through the PCP pathway can regulate their own migration, but paracrine WNT5A from macrophages can also polarize neighboring epithelial cells during tissue morphogenesis.

The Wnt/calcium pathway involves Wnt binding to Frizzled receptors, leading to activation of phospholipase C (PLC), which generates inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers calcium release from the endoplasmic reticulum, activating calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. This pathway regulates cell adhesion, migration, and inflammatory gene expression. Macrophage-derived WNT5A is a classic activator of this pathway in recipient cells, and it has been implicated in inflammatory diseases such as rheumatoid arthritis and atherosclerosis.

Regulation of Wnt Production in Macrophages

Transcriptional Control

Wnt gene expression in macrophages is regulated by a variety of transcription factors responding to microenvironmental cues. Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) induce WNT5A expression via NF-κB. The Nf Kappa B Signaling Pathway is a central mediator of this response; upon LPS stimulation, NF-κB p65/p50 heterodimers bind to the WNT5A promoter and drive transcription. Similarly, TNF-α, a cytokine that signals through Tnf Signaling Via Nf KB, can upregulate WNT2 and WNT3A in macrophages.

Other transcription factors involved include PPARγ, which promotes M2 polarization and increases WNT2 and WNT7B expression, and STAT6, which is activated by IL-4 and IL-13 and drives WNT7B transcription. Hypoxia-inducible factor 1α (HIF1α) also regulates Wnt expression in macrophages within hypoxic tumor microenvironments, often upregulating WNT5A.

Post-Translational Modifications

Post-translational regulation of Wnt proteins in macrophages is critical for controlling the amount and activity of secreted ligand. As noted, PORCN-mediated palmitoylation is essential. Pharmacological inhibition of PORCN with compounds such as LGK974 or IWP-2 abolishes Wnt secretion from macrophages in vitro and in vivo. This is a common experimental tool to test the functional contribution of macrophage-derived Wnts.

Additionally, Wnt proteins can be modified by glycosylation, which affects their folding and stability. The extracellular Wnt antagonist family—including secreted Frizzled-related proteins (sFRPs), Wnt inhibitory factor 1 (WIF1), and Dickkopf (DKK) proteins—can sequester Wnt ligands or block receptor binding. Macrophages themselves can secrete sFRP1 and sFRP2, creating a negative feedback loop that limits Wnt signaling in the local microenvironment.

Influence of Macrophage Polarization (M1/M2)

Macrophage polarization profoundly influences the Wnt ligand repertoire. M1 macrophages, induced by IFN-γ and LPS, are pro-inflammatory and predominantly express WNT5A. WNT5A activates non-canonical signaling and can amplify inflammatory responses by promoting NF-κB activity in neighboring cells. M2 macrophages, induced by IL-4, IL-13, or IL-10, are tissue-repair oriented and express WNT2, WNT7B, and WNT3A, which support canonical β-catenin signaling and promote epithelial proliferation and wound healing.

This polarization-dependent switch is not absolute; macrophages can co-express multiple Wnts, and the balance shifts with the tissue environment. In tumors, TAMs often adopt an M2-like phenotype and secrete canonical Wnts that sustain cancer stem cells. In contrast, in acute inflammation, M1-like macrophages secrete WNT5A, which can drive fibroblast activation and contribute to fibrosis if inflammation becomes chronic.

Physiological Roles in Tissue Development and Homeostasis

Role in Embryonic Patterning

During embryonic development, macrophages colonize tissues early and contribute to morphogenesis. In the developing brain, microglia (the resident macrophages) secrete Wnt ligands that support neural progenitor proliferation and differentiation. In the limb bud, macrophages produce WNT7B, which is required for proper chondrocyte maturation and bone formation. The interplay between macrophage-derived Wnts and other developmental pathways, such as the Notch Signaling Pathway, is an active area of research; both pathways often converge on shared target genes to coordinate tissue patterning.

In the developing kidney, macrophages secrete WNT4, which is essential for tubulogenesis. Genetic ablation of macrophages in mouse embryos leads to developmental defects in these organs, underscoring the non-redundant role of macrophage-derived Wnts.

Stem Cell Niche Maintenance

Adult stem cells reside in specialized microenvironments, or niches, that provide signals for self-renewal and differentiation. Macrophages are integral components of several stem cell niches. In the intestinal crypt, macrophages located near the crypt base secrete WNT2B and WNT3A, which maintain Lgr5+ intestinal stem cells. Depletion of macrophages in mice leads to impaired crypt regeneration after injury.

In the hair follicle, macrophages secrete WNT7B during the anagen (growth) phase, activating β-catenin signaling in hair follicle stem cells. In the bone marrow, macrophages support hematopoietic stem cells (HSCs) by secreting WNT3A and other niche factors. The loss of these macrophages, as occurs in certain stress conditions, leads to HSC exhaustion.

Tissue Regeneration

Macrophage-derived Wnts are critical for tissue repair after injury. In the liver, Kupffer cells secrete WNT3A and WNT2 after partial hepatectomy, driving hepatocyte proliferation. In the skin, wound macrophages produce WNT7B and WNT10A, which promote keratinocyte migration and re-epithelialization. In skeletal muscle, macrophages infiltrating injured muscle secrete WNT7A, which stimulates satellite cell proliferation and differentiation.

The timing of Wnt secretion is tightly regulated. In the early inflammatory phase, macrophages secrete WNT5A, which promotes immune cell recruitment. In the proliferative phase, the Wnt profile shifts toward canonical ligands that drive tissue growth. In the remodeling phase, Wnt secretion declines, allowing for maturation and scar resolution. Disruption of this temporal pattern can lead to fibrosis or impaired healing.

Role in Inflammation and Immune Regulation

Wnt Signaling in Macrophage Activation

Macrophages not only produce Wnts but also respond to them. Autocrine Wnt signaling in macrophages modulates their activation state. WNT5A, for example, activates the Wnt/calcium pathway in macrophages, leading to increased expression of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. This is mediated in part by CaMKII and calcineurin, which activate NF-κB and AP-1 transcription factors.

Conversely, canonical Wnt signaling in macrophages can suppress inflammation. WNT3A treatment of macrophages reduces LPS-induced TNF-α production and promotes an M2-like phenotype. This is mediated by β-catenin-dependent inhibition of NF-κB transcriptional activity. Thus, the balance between canonical and non-canonical Wnt signaling in macrophages determines their inflammatory tone.

Cross-Talk with Inflammatory Pathways

Wnt signaling intersects with multiple inflammatory pathways. As mentioned, NF-κB is both a regulator of Wnt gene expression and a downstream target of Wnt signaling. This creates a positive feedback loop in which inflammatory stimuli induce WNT5A, which then amplifies NF-κB activity. Similarly, Wnt signaling can modulate the Camp Signaling Pathway Kegg, as β-catenin can interact with cAMP-responsive element binding protein (CREB) to regulate gene expression.

Wnt signaling also cross-talks with Notch signaling. In macrophages, Notch activation can upregulate WNT5A expression, and Wnt signaling can in turn modulate Notch target genes. This cross-talk is particularly relevant in the tumor microenvironment, where both pathways are frequently activated. The Notch Delta Signaling and Notch Signaling and Neuronal Development references provide additional context on Notch pathway biology.

Implications in Cancer and Fibrosis

Tumor-Associated Macrophages (TAMs)

Tumor-associated macrophages are a major source of Wnt ligands in the tumor microenvironment. TAMs often adopt an M2-like phenotype and secrete WNT2, WNT3A, and WNT7B, which activate canonical Wnt signaling in cancer cells. This promotes cancer cell proliferation, stemness, and resistance to chemotherapy. The role of Wnt signaling in cancer is extensively covered in the Wnt Signaling in Cancer reference.

In colorectal cancer, TAM-derived WNT2 activates β-catenin signaling in tumor cells, maintaining the cancer stem cell population. In breast cancer, TAM-secreted WNT3A promotes epithelial-to-mesenchymal transition (EMT), enhancing metastatic potential. In hepatocellular carcinoma, Kupffer cell–derived WNT3A supports tumor growth and angiogenesis.

Wnt Signaling in Metastasis

Macrophage-derived Wnts also facilitate metastasis. WNT5A secreted by TAMs can activate non-canonical signaling in cancer cells, promoting cell migration and invasion. In some contexts, WNT5A also induces EMT, a process by which epithelial cancer cells acquire mesenchymal features and become motile. Additionally, macrophage-derived Wnts can remodel the extracellular matrix by activating fibroblasts, creating a permissive environment for metastatic colonization.

The pre-metastatic niche—a site where disseminated tumor cells will eventually colonize—is also influenced by macrophage Wnt signaling. Macrophages in the lung or liver can secrete Wnts that prepare the tissue for tumor cell arrival by promoting angiogenesis and suppressing anti-tumor immunity.

Fibrosis and Wnt

Fibrosis is characterized by excessive deposition of extracellular matrix components, leading to organ dysfunction. Macrophage-derived Wnts are key drivers of fibrosis in multiple organs. In the lung, alternatively activated macrophages secrete WNT5A and WNT7B, which activate fibroblasts and promote collagen production. In the kidney, macrophages produce WNT4, which drives tubular epithelial cell injury and interstitial fibrosis.

The mechanism involves Wnt-mediated activation of β-catenin signaling in fibroblasts, leading to their transdifferentiation into myofibroblasts. Myofibroblasts are the primary matrix-producing cells in fibrosis. Additionally, Wnt signaling can induce the expression of pro-fibrotic cytokines such as TGF-β, creating a feed-forward loop that amplifies fibrosis.

Experimental Approaches to Study Macrophage Wnt Signaling

In Vitro Co-Culture Systems

A common approach to study macrophage-derived Wnt signaling is co-culture. Macrophages (e.g., bone marrow-derived macrophages, BMDMs, or RAW264.7 cells) are cultured with recipient cells such as epithelial cells, fibroblasts, or stem cells. The recipient cells are then assayed for Wnt pathway activation, typically by measuring β-catenin stabilization or target gene expression.

To confirm that the effect is due to Wnt secretion, several controls are used. First, conditioned medium from macrophages can be collected and applied to recipient cells. Second, PORCN inhibitors (e.g., IWP-2 at 1–5 µM) can be added to macrophages to block Wnt secretion; if the effect on recipient cells is lost, it is Wnt-dependent. Third, neutralizing antibodies against specific Wnts or Frizzled receptors can be used to block signaling.

Genetic Mouse Models

Conditional knockout mice are essential for studying macrophage-specific Wnt production in vivo. The LysM-Cre driver is commonly used to delete genes in macrophages and neutrophils. For example, crossing LysM-Cre with WNT3A-floxed mice deletes WNT3A in macrophages, allowing researchers to assess the role of macrophage-derived WNT3A in tissue regeneration or tumor growth.

Alternatively, diphtheria toxin receptor (DTR) transgenic mice allow for inducible macrophage depletion. In these mice, administration of diphtheria toxin eliminates macrophages, and the resulting phenotype can be compared to controls. This approach has been used to show that macrophage depletion impairs intestinal stem cell maintenance and liver regeneration.

Wnt Reporter Assays

Wnt reporter cell lines are widely used to quantify Wnt activity. The most common is the SuperTOPFlash reporter, which contains multiple TCF/LEF binding sites driving firefly luciferase expression. When Wnt signaling is active, β-catenin enters the nucleus and activates the reporter. This assay is quantitative and can be used to measure the activity of macrophage-conditioned medium.

Another reporter is the TOP-GFP mouse, which expresses green fluorescent protein under the control of TCF/LEF binding sites. This allows for in vivo visualization of Wnt-active cells. However, reporter assays have limitations. They measure pathway activity, not specific ligand identity, and they can be confounded by non-Wnt signals that affect β-catenin stability.

Single-Cell Transcriptomics

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of macrophage heterogeneity and Wnt expression. By profiling individual macrophages, researchers can identify distinct subpopulations that express specific Wnt ligands. For example, scRNA-seq of tumor-associated macrophages has revealed a WNT2-expressing subpopulation that correlates with poor prognosis in colorectal cancer.

This approach also allows for the identification of ligand–receptor pairs by combining scRNA-seq data with computational tools such as CellChat or NicheNet. These tools predict cell–cell communication based on expression of ligands and receptors, providing a systems-level view of macrophage Wnt signaling in tissues.

Common Pitfalls and Misconceptions

Autocrine vs Paracrine Signaling

A frequent error is assuming that Wnt effects observed in macrophages are necessarily paracrine. Macrophages also express Frizzled receptors and can respond to their own Wnts. To distinguish autocrine from paracrine signaling, one must use conditioned medium transfer experiments or co-culture with physical separation (e.g., Transwell inserts). If the effect on recipient cells requires direct contact, it may be contact-dependent rather than soluble Wnt-mediated.

Wnt Antagonists and Modulators

Another pitfall is ignoring the role of Wnt antagonists. Macrophages secrete sFRPs and DKK proteins, which can inhibit Wnt signaling in the local environment. If a co-culture experiment shows no effect on recipient cells, it may be because the macrophages are simultaneously secreting Wnts and antagonists. Measuring the net Wnt activity with a reporter assay is essential to account for this balance.

Reporter Assay Artifacts

Wnt reporter assays are powerful but can produce false positives. For example, lithium chloride (LiCl) inhibits GSK3 and stabilizes β-catenin, activating TOPFlash without Wnt ligand. Similarly, certain growth factors can activate β-catenin through PI3K/Akt signaling. To confirm that reporter activation is Wnt-specific, one should use PORCN inhibitors or Frizzled antagonists. Additionally, some cell lines have mutations in the Wnt pathway (e.g., APC mutations in SW480 cells) that make them constitutively active; these should be avoided for measuring ligand-dependent signaling.

Frequently Asked Questions

What is Wnt signaling from macrophages?

Wnt signaling from macrophages refers to the paracrine secretion of Wnt glycoproteins by macrophages, which then activate Wnt pathways in neighboring cells. This process is important for tissue development, stem cell maintenance, immune regulation, and disease progression.

How do macrophages secrete Wnt proteins?

Macrophages synthesize Wnt proteins in the ER, where PORCN adds a palmitoleate lipid group. The lipid-modified Wnt binds to the cargo receptor WLS, which transports it to the plasma membrane for release. Secreted Wnts can diffuse locally or be carried in exosomes.

What is the difference between canonical and non-canonical Wnt signaling?

Canonical Wnt signaling stabilizes β-catenin, which enters the nucleus to activate TCF/LEF transcription factors. Non-canonical pathways, including PCP and Wnt/calcium, operate independently of β-catenin and regulate cytoskeletal dynamics, cell polarity, and calcium flux.

Do macrophages respond to Wnt signaling themselves?

Yes. Macrophages express Frizzled receptors and co-receptors. Autocrine WNT5A signaling promotes pro-inflammatory activation, while canonical WNT3A signaling can suppress inflammation and promote an M2-like phenotype.

How is macrophage Wnt signaling studied in the lab?

Common methods include co-culture assays, conditioned medium transfer, PORCN inhibitor treatment, conditional knockout mice (e.g., LysM-Cre), Wnt reporter cell lines (e.g., SuperTOPFlash), and single-cell RNA sequencing.

What is the role of macrophage Wnt signaling in cancer?

Tumor-associated macrophages secrete Wnts such as WNT2 and WNT3A that activate β-catenin signaling in cancer cells, promoting proliferation, stemness, metastasis, and chemoresistance. WNT5A from TAMs can also drive invasion via non-canonical signaling.

Can macrophage Wnt signaling be targeted for therapy?

Yes. PORCN inhibitors (e.g., LGK974) block Wnt secretion and are in clinical trials. Antibodies against specific Wnts or Frizzled receptors are also being developed. However, targeting macrophage Wnt signaling must be balanced against the essential roles of Wnts in normal tissue homeostasis.

Key Takeaways

Macrophages are significant sources of Wnt ligands that act paracrine on neighboring cells to regulate development, stem cell maintenance, and tissue repair.
Wnt secretion requires PORCN-mediated lipidation and WLS-dependent transport; blocking PORCN abolishes Wnt release.
Canonical Wnt signaling stabilizes β-catenin and activates TCF/LEF target genes, while non-canonical pathways (PCP and calcium) regulate cytoskeletal dynamics and inflammation.
Macrophage polarization dictates the Wnt repertoire: M1 macrophages predominantly secrete WNT5A, while M2 macrophages secrete WNT2, WNT3A, and WNT7B.
Macrophage-derived Wnts are critical in cancer, where TAMs promote tumor growth and metastasis, and in fibrosis, where they drive myofibroblast activation.
Experimental approaches include co-culture assays, PORCN inhibitors, conditional knockout mice, Wnt reporter cell lines, and single-cell transcriptomics.
Distinguishing autocrine from paracrine Wnt signaling and accounting for Wnt antagonists are essential for accurate interpretation of experiments.