This study suggests that inflammatory genes play a role in regulating bone mass and bone loss and influence the risk of incident fractures in elderly women. Specifically, we report association between polymorphisms in the inflammatory genes CIITA, CLEC16A and IFNG with BMD, ultrasound parameters, annual rate of bone loss and incident fractures in 75-year old women followed for 10 years. These results support inflammation and, specifically, MHCII expression as key components in postmenopausal or senile osteoporosis.
The reported association of CIITA
polymorphisms to BMD in elderly women is contrasted by the absence of association in young adult women, which suggests differential effects of these genes across the life span. It also supports our hypothesis that low-grade systemic inflammatory processes are elements of normal ageing in women, possibly due to the link between estrogen and cytokine levels. Estrogen receptors are expressed on both lymphocytes and mononuclear cells 
, making them responsive to changes in estrogen levels. Estrogen deficiency leads to increased immune activation mediated by antigen presenting cells (APCs) and cytokines (IFNγ, IL-7 and transforming growth factor-β), resulting in increased TNFα production from activated T lymphocytes 
. By increasing both the production and sensitivity to RANKL, TNFα has potent effects on osteoclasts 
. Polymorphisms affecting immune activation processes could therefore have an impact during periods with generally higher pro-inflammatory profile, such as old age and illness. We interpret the discrepant results between the elderly and young adult women as a reflection of differences in duration of exposure to low-grade systemic inflammation. The potentially deleterious effects of inflammation should be more pronounced if the systemic inflammatory processes have continued for years. This could also explain why, despite numerous GWAS of osteoporosis related phenotypes 
, these SNPs have not previously been identified; GWAS are not able to identify gene-environment interactions while the top 15 SNPs associated with BMD account for <3% of the variance of BMD 
Our observed results in elderly women - an association between functional polymorphisms in CIITA
, BMD and fracture- is supported by previous reports evaluating the impact of the MHC2TA protein on molecular interactions and pathways. MHC2TA regulation of osteoclasts has been described in murine cells where it negatively regulated NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1) and OSCAR by sequestering CBP/p300 from their promoter regions 
. Decreased expression of CIITA
would in this context lead to increased formation of osteoclasts. However, alternative mechanisms are also suggested since transgenic mice overexpressing CIITA
display a hyper-osteoclastogenic phenotype and increased activation of signaling downstream of RANK (E. Benasciutti and S. Cenci, personal communication).
Based on our observations in this study and from earlier results from us and co-workers on expression levels 
, we postulate that the studied CIITA
polymorphisms have osteoclast-stimulating effects that override the direct, inhibitory effect of IFNγ on osteoclasts 
. Mechanistically this may be explained by estrogen deficiency after menopause, leading to increased production of IFNγ 
and an induction of CIITA
expression in cells of the monocyte/macrophage lineage. The resulting MHC2TA protein acts as a transcriptional transactivator at MHCII promoters, and induces expression of MHCII molecules on antigen presenting cells 
. The T cell pool then increases due to inhibited T cell apoptosis by the increased amount of IFNγ and more T cells become activated by binding MHCII molecules. The activated T cells produce TNF, which could stimulate osteoclast activity through induction of RANKL, and increase osteoclast number through stimulating cytokines. The increase in osteoclast activity could then lead to decreased BMD and increased risk of fracture. The observed protective effect from the CIITA
rs3087456(G) allele is associated with lower expression of CIITA
and MHCII 
, which could slow down the process of T cell- and osteoclast stimulation and result in higher BMD and reduced fracture risk.
In the present study, the inverse effect of CIITA variant alleles on rate of bone loss compared to BMD and fracture is however somewhat contradictory. Individuals carrying CIITA variant alleles (mainly CIITA rs3087456(G)) lost more bone mineral density between age 75 and 80, but had higher BMD at age 75, 80 and 85 and, importantly, were protected from incident fracture during this time. CIITA variant alleles thus had a net positive effect on bone density and bone strength in elderly women at all time-points, despite also being associated with a higher rate of bone loss. Gene effects on bone turnover were not reflected by the bone resorption markers S-CTX and S-TRACP5b, however, the low numbers with consistently high or low turnover may have masked such effects. Furthermore, markers indicate turnover (and the potential effect of inflammatory processes on it) only at the time of sample collection and may not be able to capture the long-term effects of inflammation.
In addition to CIITA, CLEC16A and to a lesser extent INFG were associated with bone mass phenotypes. However, the contribution to fracture risk is less clear; CLEC16A was associated with a significantly reduced fracture risk, although less than CIITA, while INFG was not. A potential explanation would be that the CLEC16A marker most strongly associated with fracture (rs725613), is located closest to, and is in weak LD with CIITA.
The strengths of the study include the comprehensive and extended evaluation of the women in the OPRA-cohort, including detailed fracture information and bone turnover markers. We also have the possibility to make direct comparisons of ageing effects through the large cohort of young adult women in the PEAK-25 cohort. In this study, the reported association with bone phenotypes and overall fracture risk in the elderly women support the relevance of inflammatory genes affecting MCHCII expression in the pathogenesis of osteoporosis.
The study has some limitations, notably the large number of tests performed. Taking this into consideration and applying a conservative Bonferroni correction, the results can be considered to fall short of p<0.05. Performing adjustment at least for the 8 markers tested, association with BMD is non-significant, whereas association of CIITA with total body RBL, fracture and hip fracture is still significant. Importantly however the direction of effect of the SNPs within each gene is consistent indicating biological plausibility for the observations and importantly, both cohorts are of sufficient size to be confident of detecting true genotype related differences in the phenotypes analysed.
Replication studies are needed in order to fully evaluate the effect of the studied SNPs on osteoporosis-related phenotypes. Thus, replication in a similarly aged cohort to OPRA is necessary to corroborate the findings. Secondly, routine biochemistry including leukocyte count, CRP and ESR as clinical indicators of inflammation was only available on all OPRA participants at age 85. The finding that CLEC16A
haplotypes were associated with higher counts of blood leukocytes with a similar trend for single markers, suggests that it would have been interesting to have complete longitudinal data. Thirdly, longitudinal data for the young adult women would be potentially interesting (including follow-up extending into the perimenopausal years) in order to facilitate evaluation of the natural course of inflammatory influence with normal ageing and transition to an estrogen deplete state. In a study comparing levels of IFNγ in young, perimenopausal and elderly women, the levels of IFNγ increased at menopause compared to young women, but fell to even lower levels in the elderly 
. These results strongly argue for testing the studied CIITA
polymorphisms in cohorts of perimenopausal women for comparison.
We conclude that expression-related polymorphisms in the inflammatory genes CIITA and CLEC16A are associated with BMD and fracture in elderly women. These findings illustrate the importance of inflammation and MHCII expression in particular in the pathogenesis of reduced bone strength in the elderly.