11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an
enzyme that predominantly converts inactive glucocorticoids (cortisone in human
and most mammals, 11dehydro-corticosterone in mice and rats) into their active
forms (cortisol and corticosterone, respectively). Thus 11β-HSD1 amplifies intracellular
levels of glucocorticoids. Studies in globally 11β-HSD1 deficient mice have
revealed changes in glucocorticoid-regulated physiological and pathological
processes, including metabolism, aging, arthritis and angiogenesis. The function of
macrophages, which play an important role in inflammation, is also altered. For
example, 11β-HSD1 deficiency in macrophages causes a delay in their acquisition of
phagocytic capacity. To dissect the role of macrophage 11β-HSD1 in angiogenesis,
arthritis and obesity, both in vitro macrophage stimulation and in vivo functional
assays in macrophage-specific 11β-HSD1 knockout mice, were conducted.
Thioglycollate-elicited peritoneal macrophages from globally 11β-HSD1 deficient
and control C57BL/6 mice were used for in vitro studies. In M1/M2 macrophage
polarisation experiments, 11β-HSD1 deficient macrophages showed increased
expression of mRNAs encoding pro-inflammatory factors upon lipopolysaccharide
and interferon-ϒ treatment and decreased expression of pro-resolution genes with
interleukin-4 stimulation. However, at cytokine or protein levels, there was little
difference between the genotypes except for decrease IL12 p40 levels in 11β-HSD1
deficient macrophages. Hypoxic stress failed to show differences between genotypes
in hypoxia-regulated gene expression. These data do not support a strong role for
macrophage 11β-HSD1 in inflammation regulation, nor in response to hypoxia, at
least when measured in vitro. The discrepancy between transcriptional and
translational responses is currently unexplained, but may reflect altered posttranscriptional
activity.
To investigate the role of macrophage 11β-HSD1 in vivo, macrophage-specific
Hsd11b1 knockout mice, LysM-Cre Hsd11b1 flox/flox (MKO) mice and Hsd11b1flox/flox littermate controls were generated. In MKO mice, 11β-HSD1 protein levels and
enzyme activity were reduced by >80% in resident peritoneal macrophages. However,
11β-HSD1 protein and enzyme activity levels were unchanged or only modestly
reduced in thioglycocollate-elicited peritoneal neutrophils, monocytes/macrophages,
or in bone marrow-derived macrophages, despite >80% decrease in Hsd11b1 mRNA
levels in these cells. A relatively long half-life of 11β-HSD1 protein compared to that
of circulating myeloid cells may underlie this mismatch between transcriptional and
translational expression. Furthermore, following 12 days of inflammatory arthritis
induced by K/BxN serum transfer, the reduction in 11β-HSD1 protein levels in
circulating neutrophils of MKO mice is consistently around 50%, which corroborates
the above explanation.
MKO mice and littermate controls were subjected to inflammatory models which
may involve resident macrophages. First, to address the role of 11β-HSD1 in
macrophages in angiogenesis, sponge implants were inserted subcutaneously into the
flanks of adult male mice and harvested after 21 days. Chalkley counting on
hematoxylin and eosin stained sponge sections showed significantly increased
angiogenesis in MKO mice (scores: 5.2±1.0 versus 4.3±0.7; p<0.05, n=9-11). Cdh5
expression (encoding VE-cadherin, a marker of endothelial cells) was higher in
sponges from MKO mice (relative expression: 1.5±0.9 versus 0.8±0.6; p<0.05), as
was Il1b (encoding IL-1 beta, a marker of inflammation, relative expression: 6.5±6.4
versus 1.5±0.9; p<0.05). Vegfa mRNA (encoding vascular endothelial growth factor
alpha) was unchanged, with a trend for higher Angpt1 (encoding angiopoietin 1,
p=0.09) expression levels in the MKO group. These results suggest that lack of 11β-
HSD1 in resident macrophages increases their pro-angiogenic activity, independently
of VEGF-.
The K/BxN serum transfer model of arthritis was used to investigate the role of
macrophage 11β-HSD1 in arthritis. Adult male MKO and control mice received a
single i.p. injection of 125μl K/BxN serum per mouse, followed by 21 days of
clinical scoring to assess joint inflammation. The onset of inflammation (d1-8) was
similar between MKO and control mice, but MKO mice exhibited greater clinical inflammation scores in the resolution phase of arthritis (d13-21; area-under-the-curve:
86.6±14.7 versus 60.1±13.4; p<0.005), indistinguishable from globally 11β-HSD1-
deficient mice. Hematoxylin and eosin staining revealed pronounced fibroplasia
predominantly in the supporting mesenchyme associated with the tenosynovium,
with new bone and blood vessel formation. These results suggest that macrophage
11β-HSD1 deficiency is fully accountable for the worse arthritis resolution
phenotype in the globally 11β-HSD1 deficient mice, but not the earlier onset of
inflammation with global 11β-HSD1 deficiency. Macrophage activation states are closely linked with adipose insulin sensitivity.
Globally 11β-HSD1 deficient mice are protected from high fat diet induced insulin
resistance and adipose tissue hypoxia and fibrosis. To study the effect of macrophage
11β-HSD1 deficiency on insulin sensitivity, adult male MKO and control mice were
given a 14 week high fat diet, which typically causes insulin resistance in control but
not globally 11β-HSD1 KO mice. The level of fibrosis in subcutaneous adipose
tissues was reduced as indicated by quantification of picrosirius red staining of
collagen, though GTT data so far does not support protection from insulin resistance
in MKO mice.
In summary, in vitro macrophage polarisation experiments do not support a strong
role of 11β-HSD in M1/M2 macrophage polarisations or response to hypoxia.
However, MKO mice reveal, for the first time, an important in vivo role of
macrophage 11β-HSD1 to promote angiogenesis and facilitate resolution of K/BxN
serum transfer induced arthritis. Modulation of fibrosis is context dependent.
Reduced adipose fibrosis may be one of the mechanisms that improve insulin
sensitivity. Meanwhile, these findings suggest caution regarding the potential side
effects of 11β-HSD1 inhibitors in treating metabolic disease in patients with
inflammation-related co-morbidities, such as rheumatoid arthritis