Similarities and differences between IL11 and IL11RA1 knockout mice for lung fibro-inflammation, fertility and craniosynostosis

Loss of function (LOF) in IL11RA infers IL11 signaling as important for fertility, fibrosis, inflammation and incompletely penetrant craniosynostosis. The impact of LOF in IL11 has not been characterized. We generated IL11 knockout (Il11−/−) mice that are born in expected ratios and have normal hematological profiles. Lung fibroblasts from Il11−/− mice are resistant to pro-fibrotic stimulation with TGFβ1. Following bleomycin-induced lung injury, Il11−/− mice are protected from pulmonary fibrosis and exhibit lesser ERK, STAT3 and NF-kB activation, reduced Il1b, Timp1, Ccl2 and diminished IL6 expression, both at baseline and after injury: placing Il11 activity upstream of IL6 in this model. Il11−/− female mice are infertile. Unlike Il11ra1−/− mice, Il11−/− mice do not have craniosynostosis, have normal long bone mass and reduced body weights. These data further establish the role of IL11 signaling in lung fibrosis while suggesting that bone development abnormalities can be associated with mutation of IL11RA but not IL11, which may have implications for therapeutic targeting of IL11 signaling.


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Interleukin 11 (IL11) was originally described as a factor important for hematopoiesis, 38 notably platelet production, but more recently found to drive fibro-inflammatory 39 disorders 1 . IL11 is a member of the IL6 family of cytokines, which share the gp130 40 coreceptor, but while IL6 has been studied in very great detail with an armamentarium 41 of genetic tools to dissect its function, IL11 remains poorly characterised 1 .

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It is apparent from the published literature that the majority of our understanding 44 of the biology associated with loss-of-function (LOF) in IL11 signaling is inferred from 45 studies of IL11RA mutant humans or mice 1 . The field of lL11 biology has lacked a 46 mouse genetic model specific for IL11 LOF, which represents a gap in our 47 understanding. This is important as, in the case of the family member IL6, there are 48 both similarities and differences between effects of LOF in the IL6 cytokine as 49 compared to LOF in its cognate receptor alpha chain (IL6RA) 2,3 . As such, it is possible 50 that the phenotype of IL11RA LOF may not map precisely to IL11 function. Furthermore, 51 studies of IL11RA1 LOF have been conducted in a single mouse strain and there are 52 additional genes in the targeted locus, which is a potential shortcoming.

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Based on the genetic studies of IL11RA mutants, IL11 signaling is thought 55 important for a number of phenotypes. Il11ra1-deleted female mice are infertile 4 and 56 mutation in Il11ra1 in the mouse is associated with incompletely penetrant snout 57 Deletion of Il11ra1 in mice leads to female infertility due to defective embryo 102 implantation related to abnormal placental decidualization, whereas Il11ra1 -/males are 103 fertile 4,11,12 . Intercrosses of heterozygotes (Il11 +/-) gave rise to viable and apparently 104 normal homozygous mutant (Il11 -/-) mice in the expected Mendelian ratios ( Fig. 2A). We 105 determine whether maternal Il11 expression is required for fertility by mating 106 homozygous (Il11 -/-) female mice with male mice of variable IL11 genotype ( Il11 +/+ , 107 Il11 +/or Il11 -/-) and found that female mice deficient for Il11 never had a detectable 108 pregnancy nor gave birth to offspring (Fig. 2B), which mirrors the infertility phenotype of 109 homozygous Il11ra1 -/female mice 4 . Crossing homozygous (Il11 -/-) male mice with 110 either wild-type (Il11 +/+ ) or heterozygous (Il11 +/-) female mice resulted in viable offspring 111 of expected Mendelian ratios. However, litter sizes derived from Il11 -/male mice were 112 significantly smaller as compared to intercrosses of heterozygotes (Fig. 2C). Hence, 113 similar to germline loss of Il11ra1, the loss of Il11 expression results in female infertility 114 and appears to affect male fertility, directly or indirectly, in mice.

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Blood hematology and chemistry profiles are normal in Il11-knockout mice. 117 We evaluated the hematological profile of adult Il11 -/mice (10-14 weeks of age) and 118 observed that null mice had normal peripheral red and white blood cell counts as well as 119 normal platelet counts and volumes as compared to wild-type mice ( Table 1). Likewise, 120 we profiled serum chemistry and observed normal levels of serum markers of liver 121 function (albumin, alanine aminotransferase, total bilirubin), kidney function (blood urea 122 nitrogen, sodium and potassium) and bone turnover (alkaline phosphatase, calcium and 123 phosphate) in Il11 -/mice ( Table 1). These data indicate that Il11 -/mice have normal 124 blood hematological and chemistry profiles under normal physiological conditions. 125 126 IL11 is required for myofibroblast differentiation. 127 We previously found that TGFβ1-induced myofibroblast transdifferentiation was 128 impaired in Il11ra1 -/lung fibroblasts 13 . To examine whether the loss of the endogenous 129 IL11 autocrine feed-forward loop similarly perturbed fibroblast activation, we stimulated 130 lung fibroblasts from Il11 -/mice with recombinant mouse TGFβ1 or IL11 (5 ng/ml; 24 131 hours) and monitored fibroblast activation using automated high-throughput 132 immunofluorescence imaging and Sirius red-based quantification of secreted collagen. 133 In keeping with the data from Il11ra1-deleted fibroblasts, the differentiation of Il11 -/-134 fibroblasts into ACTA2 +ve and COL1A1 expressing myofibroblasts following TGFβ1 135 stimulation was significantly diminished (Fig. 3A-B). Cell proliferation (as determined by 136 EdU +ve staining) and secreted collagen levels into the culture supernatant were also 137 significantly reduced in Il11 -/fibroblasts following TGFβ1 stimulation (Fig. 3C-D).

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We next addressed whether disruption of the IL11 locus prevented IL11 protein 140 expression by performing ELISA on culture supernatants and found that IL11 protein 141 was not expressed by Il11 -/lung fibroblasts at baseline or after TGFβ1 stimulation (Fig.   142   3E). Interestingly, recombinant mouse IL11 (5ng/ml) did not fully restore pro-fibrotic 143 phenotypes in Il11 -/fibroblasts ( Fig. 3A-D). We assessed the expression of IL11RA in 144 Il11 -/lung fibroblasts by immunofluorescence staining and detected comparable levels 145 of IL11RA expression between Il11 -/and wild-type cells (Fig. 3F). This suggests that 146 the autocrine loop of IL11 in the local environs of the cell is of greater importance for 147 pro-fibrotic activity than exogenous IL11.

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Bleomycin-induced pulmonary fibrosis is attenuated in Il11-knockout mice. 150 We recently showed that IL11 expression is elevated in the lung after bleomycin (BLM)- that Il11 -/mice had reduced parenchymal disruption and fibrosis ( Fig. 4C-D). These 158 changes were associated with significantly lower total lung hydroxyproline (collagen) 159 content in Il11 -/mice (Fig 4E). and Timp1 in BLM-challenged Il11 -/mice as compared to wild-type mice ( Fig. 4F-J). 164 Reduced expression of several inflammatory response genes (such as Il1b, Il6 and 165 Ccl2) were also seen in the lungs of Il11 -/mice following BLM injury ( Fig. 4K-M). 166 167 Western blot analysis showed that IL11 protein expression was strongly 168 upregulated in the lungs of BLM-injured wild-type mice and was not expressed at all in 169 the lungs of Il11 -/mice at all, as expected (Fig. 5A). Furthermore, in BLM-treated Il11 -/-170 mice, lung protection was accompanied by reduced pulmonary fibronectin and IL6 171 protein expression (Fig. 5A) and reduced ERK activation (Fig. 5B). Notably, lung IL6 172 levels in Il11 -/mice were lower than wild type control levels in the absence of lung There is variation in reported fertility phenotypes associated with LOF mutations 224 in the IL11 pathway in mice and humans ( Table 2). Female mice lacking IL11RA1 are 225 infertile due to defective decidualization in response to embryo implantation 4,12 . In 226 contrast, women with homozygous IL11RA variants appear able to reproduce 8 which 227 could be explained by species differences in decidualization 21 . In this study, we found 228 that Il11 -/female mice are infertile, consistent with a recent report 22 . Interestingly, we