Picture a bit of bacterial membrane floating about the brain. When a patrolling microglia cell notices that, it prods neighboring microglia to prick up their ears, too. Researchers now report that their clarion call consists of a protein called galectin-3, which binds toll-like receptor 4 (TLR4) on other microglia. In the March 10 Cell Reports, first author Miguel Burguillos of Queen Mary University in London, and colleagues also report that galectin-3 and TLR4 abound in the brains of people who suffered ischemia, suggesting the same alert system could be active in humans after loss of blood flow to the brain. As for neurodegenerative conditions, galectin-3 is known to flood the spinal cords of people with amyotrophic lateral sclerosis. Researchers are aware of a powerful neuroinflammatory component to several neurodegenerative diseases, but have not yet analyzed galectin-3 in detail (see Feb 2015 news; news story; Jul 2014 news).

Wake-Up Call.

In response to a loss of blood to the brain, microglia secrete galectin-3 to activate their neighbors. [Courtesy of Cell Reports, Burguillos et al.]

Galectins: Sweet-Toothed, Two-Faced 
A family of sugar-binding proteins, galectins interact with carbohydrates on other molecules, such as glycoproteins. They operate inside cells and in the extracellular space, and participate in a variety of processes, including proliferation, neurite growth, and apoptosis. Galectins can take on opposing roles depending on their location and circumstances. For example, galectin-1 modulates binding of cells to the extracellular matrix; promoting this binding in melanoma cells but inhibiting it in muscle (reviewed in Cooper and Barondes, 1999). 

Scientists have long been aware that galectin-3 participates—somehow—in inflammation, Burguillos said. In fact, researchers use it as a marker for activated microglia and macrophages, though in that context it is often known by another name, Mac-2. Galectin-3 can be found in the nucleus, cytosol, and cellular membranes, and the cell exports it in response to inflammatory stimuli such as lipopolysaccharide, a component of bacterial membranes, or interferon-γ (Li et al., 2008; Jeon et al., 2010). 

Galectin-3 seems to either promote or prevent inflammation, depending on its situation. For example, in a mouse model of ALS, knocking out the gene enhanced neuroinflammation and sped up the disease, suggesting galectin-3 must be anti-inflammatory (Lerman et al., 2012). However, in a project on mice with experimental autoimmune encephalomyelitis, loss of galectin-3 alleviated disease, suggesting pro-inflammatory ability (Jiang et al., 2009). 

Burguillos focused on the secreted, extracellular form of galectin-3 released in response to LPS or ischemia. He worked in the laboratories of co-senior authors Bertrand Joseph of the Karolinska Institute in Stockholm and Tomas Deierborg of Lund University in Sweden. In collaboration with co-senior author Jose Venero of the University of Seville, Spain, they found a pro-inflammatory role for galectin-3 in this context.

Inflammation Enhancer 
Previous studies indicated that galectin-3 could bind to toll-like receptors as well as to LPS, which instigates inflammation via TLR4 (Mey et al., 1996). Therefore, the authors asked if galectin-3 itself might also interact with TLR4. In the mouse microglial BV2 cell line, extracellular galectin-3 co-localized with TLR4, and the two proteins co-immunoprecipitated. Burguillos measured expression of immune-related genes in the galectin-3-treated cells, and observed a pro-inflammatory profile.

Burguillos reasoned that if galectin-3 mediates inflammation, then blocking the protein should dampen microglial responses to LPS. In BV2 cultures, neutralizing galectin-3 with either interfering RNA or antibodies reduced production of nitric oxide synthase, a marker for inflammation. Experiments with primary microglia corroborated these results. 

All told, the findings suggest that secreted galectin-3 can amplify inflammation. Would this hold up in vivo? Burguillos examined galectin-3 activity in two different mouse models of neuroinflammation. First, he created a situation somewhat akin to Parkinson’s disease by injecting LPS into the substantia nigra. Galectin-3 and TLR4 co-localized in cells expressing the microglial marker Iba1, macrophages gathered near the injection site, and half the dopaminergic neurons died. In galectin-3 knockout mice, fewer macrophages were recruited and the neurons survived.

Second, Burguillos modeled ischemia. Occluding the carotid arteries for 13 minutes caused a rise in populations of Iba1-positive microglia and death of pyramidal neurons in the hippocampus of wild-type mice. Again, galectin-3 knockouts were protected.

Finally, Burguillos examined brain tissue samples from five people who had died of cardiac arrest, which would limit blood flow to the brain. These samples contained more galectin-3 and TLR4 than brains from age-matched controls.

The authors conclude that galectin-3 release starts a chain reaction of inflammation by activating other microglia. They suspect it acts only on nearby cells—microglia and perhaps astrocytes or monocytes—in a paracrine manner. Cells take up galectin-3 quickly, Burguillos pointed out, so it would probably not reach far-flung microglia. The human data suggest, but by no means prove, that a similar process occurs in people, he said.

Mixed Signals
Though Burguillos studied ischemia, he speculated that galectin-3 might be involved in neuroinflammation in other conditions where TLR4 has been implicated, including Alzheimer’s, Parkinson’s, and ALS (Chen et al., 2012; Noelker et al., 2013; Casula et al., 2011). What exactly do the new data mean for those conditions? “It is hard to decide,” said Stanley Appel of the Methodist Neurological Institute in Houston, citing the disparate pro- or anti-inflammatory roles of galectin-3 in different disease models. Appel speculated that galectin-3 might affect inflammation via receptors other than TLR4 in different conditions. Burguillos pointed out that he found a pro-inflammatory role for extracellular galectin-3, but the intracellular version could conceivably do the opposite, and this galectin-3 might contribute to the biology of other diseases.

In the case of ALS, galectin-3’s role remains in question, but it does seem to be involved. Scientists have reported its upregulation in the microglia and muscles of SOD1 rats, cerebrospinal fluid of people with ALS, and spinal cords of people who died of the disease, leading to the suggestion it could be a biomarker (Nikodemova et al., 2014; Gonzalez de Aguilar et al., 2008; Zhou et al., 2010).

“Galectin-3 may play a role in ‘propagating’ proinflammatory microglial responses,” commented Mariko Bennett of the Stanford University School of Medicine, who was not involved in the work, in an email to Alzforum (see full comment below). “While further work is needed to demonstrate the paracrine nature of these effects … perhaps this system represents a mechanism by which small amounts of injury or inflammation can lead to widespread, prolonged, and possibly neurotoxic microglia activation in neurodegenerative disease.”

However, Oleg Butovsky of Brigham and Women’s Hospital in Boston, who also was not involved in the study, questioned the cell culture experiments. He cautioned that that the Iba1 marker the authors used to identify microglia also occurs in peripheral monocytes, which usually express more galectin-3 than microglia do,  and that neither BV2 cells nor microglia from juvenile mice express the same pattern of genes as adult microglia. He said he would like to see the experiments repeated in microglia from adult mice (Butovsky et al., 2014). Burguillos concurred that cultures from adult mice would strengthen his argument.—Amber Dance

Comments

  1. In this publication, Burguillos and colleagues have identified Galectin-3 (Gal3) as an endogenous ligand for Toll-like Receptor 4 (TLR4). The paper focuses on the microglia’s inflammatory response and the authors evaluated Gal3-mediated pro-inflammatory responses in the brain under conditions of acute brain inflammation. They observed neuroprotective and anti-inflammatory effects of Gal3 depletion following global brain ischemia and in the neuroinflammatory lipopolysaccharide model. They suggest that the inflammatory role of Gal3 in the brain may differ, depending on the specific neuroinflammatory conditions. Accordingly, a previous study (Lerman et al., 2012) showed that the deletion of Gal3 exacerbates microglial activation and accelerates disease progression and demise in an SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Thus the expression pattern and the biological effect of Gal3 in different neurological disorders characterized by activation of the TLR4 signaling pathway (such as epilepsy, ALS, and ALZ), should be further clarified using both experimental models and human tissue studies. Evaluation of the role of galectins as TLR4 ligands in different model and cell types (i.e. astrocytes) also requires further investigation.

    References:

    . Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Brain Behav. 2012 Sep;2(5):563-75. PubMed.

    View all comments by Eleonora Aronica
  2. I think this is a tantalizing study for two reasons. First, it demonstrates that Galectin-3 can directly interact with Toll-like Receptor 4 (TLR4). Second, since Gal3 is generally expressed and released exclusively by microglia within the brain following inflammation, Gal3 may play a role in “propagating” pro-inflammatory microglial responses by binding TLR4 on neighboring microglia. In a mouse model of Parkinson's disease (intrastriatal lipopolysaccharide injection), dopaminergic neurons were largely spared in Gal3 knockout mice, suggesting that normally Gal3 works to promote cell death in response to LPS. Given the authors' in vitro work, one might hypothesize that Gal3 works as an accelerator for widespread microglial activation in response to LPS. While further work is needed to conclusively demonstrate the paracrine nature of these effects (mixing experiments or mice in which Gal3 is not secreted), its implications are very interesting: Perhaps this system represents a mechanism by which small amounts of injury or inflammation can lead to widespread, prolonged, and possibly neurotoxic microglial activation in neurodegenerative diseases.

    View all comments by Mariko Bennett

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References

News Citations

  1. Microglia in Disease: Innocent Bystanders, or Agents of Destruction?
  2. Neuroinflammation Field Grapples With Complexity at Keystone Symposia
  3. Inflammation in Midlife May Presage Cognitive Decline

Paper Citations

  1. . God must love galectins; he made so many of them. Glycobiology. 1999 Oct;9(10):979-84. PubMed.
  2. . Galectin-3 is a negative regulator of lipopolysaccharide-mediated inflammation. J Immunol. 2008 Aug 15;181(4):2781-9. PubMed.
  3. . Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway. J Immunol. 2010 Dec 1;185(11):7037-46. Epub 2010 Oct 27 PubMed.
  4. . Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Brain Behav. 2012 Sep;2(5):563-75. PubMed.
  5. . Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis. J Immunol. 2009 Jan 15;182(2):1167-73. PubMed.
  6. . The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites. J Immunol. 1996 Feb 15;156(4):1572-7. PubMed.
  7. . Sequence variants of toll like receptor 4 and late-onset Alzheimer's disease. PLoS One. 2012;7(12):e50771. PubMed.
  8. . Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease. Sci Rep. 2013;3:1393. PubMed.
  9. . Toll-like receptor signaling in amyotrophic lateral sclerosis spinal cord tissue. Neuroscience. 2011 Apr 14;179:233-43. PubMed.
  10. . Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats. Neurobiol Dis. 2014 Sep;69:43-53. Epub 2013 Nov 19 PubMed.
  11. . Gene profiling of skeletal muscle in an amyotrophic lateral sclerosis mouse model. Physiol Genomics. 2008 Jan 17;32(2):207-18. PubMed.
  12. . Galectin-3 is a candidate biomarker for amyotrophic lateral sclerosis: discovery by a proteomics approach. J Proteome Res. 2010 Oct 1;9(10):5133-41. PubMed.
  13. . Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014 Jan;17(1):131-43. Epub 2013 Dec 8 PubMed.

Further Reading

Papers

  1. . Caspase signalling controls microglia activation and neurotoxicity. Nature. 2011 Apr 21;472(7343):319-24. PubMed.
  2. . Different mechanisms of apolipoprotein E isoform-dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia. FASEB J. 2015 May;29(5):1754-62. Epub 2015 Jan 15 PubMed.
  3. . Regulation of alternative macrophage activation by galectin-3. J Immunol. 2008 Feb 15;180(4):2650-8. PubMed.
  4. . Toll-like receptors in Alzheimer's disease: a therapeutic perspective. CNS Neurol Disord Drug Targets. 2014;13(9):1542-58. PubMed.
  5. . Microglial phenotypes and toll-like receptor 2 in the substantia nigra and hippocampus of incidental Lewy body disease cases and Parkinson's disease patients. Acta Neuropathol Commun. 2014 Aug 7;2:90. PubMed.

Primary Papers

  1. . Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation. Cell Rep. 2015 Mar 4; PubMed.