**4. T-cell subsets and their action on osteoclasts**

#### **4.1. Th17 cells versus Th1 and Th2 cells**

The Th17 lineage has only recently been fully characterized (Adamopoulos and Bowman 2008) and the factors involved in its differentiation are still being identified and sorted out (Hirota, Hashimoto et al. 2007; Parsonage, Filer et al. 2008). When a (CD4+) naïve T cell is activated in the presence of transforming growth factor beta (TGFβ) plus IL-6 in the mouse or TGFβ plus an inflammatory stimulus in the human, the resulting clonal memory T-cell population will be instructed to produce the Th17 signature cytokines IL-17A, IL-17F, IL-22, and (only in humans) IL-26 (Manel, Unutmaz et al. 2008). The inflammatory stimuli in the human setting can be IL-1β, IL-6, IL-21, and/or IL-23 (Langrish, Chen et al. 2005; Acosta-Rodriguez, Napolitani et al. 2007; Wilson, Boniface et al. 2007; Manel, Unutmaz et al. 2008; Volpe, Servant et al. 2008; Yang, Anderson et al. 2008). IL-17A is the only Th17 signature cytokine presently known to impact osteoclast biology. Synovial residing and synovial fluid derived macrophages are able to differentiate to fully functional bone-resorbing osteoclasts, and Th17- induced synovial macrophage–mediated osteoclast differentiation is believed to represent an important mechanism in bone destruction associated with rheumatoid arthritis (Fujikawa, Sabokbar et al. 1996; Adamopoulos, Sabokbar et al. 2006). Several types of IL-17A antagonists have been applied in a variety of experimental arthritis models to elucidate the efficacy of therapeutic IL-17A neutralization. Polyclonal anti-IL-17A antibody treatment after disease induction in the collagen induced arthritis (CIA) model diminished clinical scores subsequent to 10 days of therapy, compared with controls. Ankle and knee joints displayed reduced synovitis, cartilage destruction, chondrocyte cell death, depletion of proteoglycans, and bone erosion (estimated by both histology and X-ray analyses) (Lubberts, Koenders et al. 2004). Polyclonal anti-IL-17A antibodies also suppressed exacerbation of antigen-induced knee swelling, proteoglycan depletion, and bone erosion in experimental RA model systems upon reintroduction of the antigen (Koenders, Lubberts et al. 2005). The Th1 and Th2 cell lineages, of which the Th1 cell phenotype resembles that of Th17 cells, have interestingly proven to counteract the effect of Th17 cells on osteoclastogenesis, both *in vitro* and in experimental and idiopathic conditions of rheumatoid arthritis (Aarvak, Chabaud et al. 1999).

#### **4.2. T-helper 17 cells in Rheumatoid Arthritis (RA)**

A multitude of experimental reports point to Th17 cell mediated inflammation being associ‐ ated with RA. IL-17A protein is located in both the synovium and the synovial fluid of pa‐ tients suffering from the disease. It was also demonstrated that a subset of T-cell lines could be expanded *in vitro* from their RA synovium, which expresses both IL-17A and IFNγ [(Aar‐ vak, Chabaud et al. 1999; Pene, Chevalier et al. 2008). Classical IFNγ-only Th1 cells were al‐ so present, constituting a clonal species which can be expanded from the RA synovium (Aarvak, Chabaud et al. 1999; Yamada, Nakashima et al. 2008). Exploratory medical studies, however, not only establish the association of IL-17A with disease development, but also implicate IL-17A with poor disease prognosis. The IL-17A message in synovial membrane biopsies was one factor (including TNF, IL-1β, and IL-10) being predictive for subsequent bone erosion and joint damage, as assessed by NMR imaging and X-ray analyses (Kirkham, Lassere et al. 2006). Taken together, these data support the notion that IL-17A is present in the inflamed synovium and that the levels of IL-17A expression correlate with poor progno‐ sis and greater joint destruction. All in all, Th17 cells secrete master factors that can directly and indirectly drive osteoclast differentiation and activation. It is, however, a consensus in the field of T-cell research that signature cytokines of the Th1, Th2, and Treg lineages inhibit Th17 cell development. Therefore, one has construed that osteoclastogenesis is inhibited by the same Th1, Th2, and Treg lineage signature cytokines counteracting the stimulatory po‐ tential of the cytokine mixture secreted by the Th17 cells (Adamopoulos, Sabokbar et al. 2006; Adamopoulos and Bowman 2008). Contrastingly, when comparing clonal cells lines, Th1 cells may resemble Th17 cells, questioning the postulate that the Th1 cell lineage consis‐ tently will counteract the impact of Th17 cells. In some instances, infiltration by Th1 cells may even potentiate the detrimental effect of Th17 cells on the cartilage and bone in affected joints (Yssel et al., unpublished data).

and (only in humans) IL-26 (Manel, Unutmaz et al. 2008). The inflammatory stimuli in the human setting can be IL-1β, IL-6, IL-21, and/or IL-23 (Langrish, Chen et al. 2005; Acosta-Rodriguez, Napolitani et al. 2007; Wilson, Boniface et al. 2007; Manel, Unutmaz et al. 2008; Volpe, Servant et al. 2008; Yang, Anderson et al. 2008). IL-17A is the only Th17 signature cytokine presently known to impact osteoclast biology. Synovial residing and synovial fluid derived macrophages are able to differentiate to fully functional bone-resorbing osteoclasts, and Th17- induced synovial macrophage–mediated osteoclast differentiation is believed to represent an important mechanism in bone destruction associated with rheumatoid arthritis (Fujikawa, Sabokbar et al. 1996; Adamopoulos, Sabokbar et al. 2006). Several types of IL-17A antagonists have been applied in a variety of experimental arthritis models to elucidate the efficacy of therapeutic IL-17A neutralization. Polyclonal anti-IL-17A antibody treatment after disease induction in the collagen induced arthritis (CIA) model diminished clinical scores subsequent to 10 days of therapy, compared with controls. Ankle and knee joints displayed reduced synovitis, cartilage destruction, chondrocyte cell death, depletion of proteoglycans, and bone erosion (estimated by both histology and X-ray analyses) (Lubberts, Koenders et al. 2004). Polyclonal anti-IL-17A antibodies also suppressed exacerbation of antigen-induced knee swelling, proteoglycan depletion, and bone erosion in experimental RA model systems upon reintroduction of the antigen (Koenders, Lubberts et al. 2005). The Th1 and Th2 cell lineages, of which the Th1 cell phenotype resembles that of Th17 cells, have interestingly proven to counteract the effect of Th17 cells on osteoclastogenesis, both *in vitro* and in experimental and

idiopathic conditions of rheumatoid arthritis (Aarvak, Chabaud et al. 1999).

A multitude of experimental reports point to Th17 cell mediated inflammation being associ‐ ated with RA. IL-17A protein is located in both the synovium and the synovial fluid of pa‐ tients suffering from the disease. It was also demonstrated that a subset of T-cell lines could be expanded *in vitro* from their RA synovium, which expresses both IL-17A and IFNγ [(Aar‐ vak, Chabaud et al. 1999; Pene, Chevalier et al. 2008). Classical IFNγ-only Th1 cells were al‐ so present, constituting a clonal species which can be expanded from the RA synovium (Aarvak, Chabaud et al. 1999; Yamada, Nakashima et al. 2008). Exploratory medical studies, however, not only establish the association of IL-17A with disease development, but also implicate IL-17A with poor disease prognosis. The IL-17A message in synovial membrane biopsies was one factor (including TNF, IL-1β, and IL-10) being predictive for subsequent bone erosion and joint damage, as assessed by NMR imaging and X-ray analyses (Kirkham, Lassere et al. 2006). Taken together, these data support the notion that IL-17A is present in the inflamed synovium and that the levels of IL-17A expression correlate with poor progno‐ sis and greater joint destruction. All in all, Th17 cells secrete master factors that can directly and indirectly drive osteoclast differentiation and activation. It is, however, a consensus in the field of T-cell research that signature cytokines of the Th1, Th2, and Treg lineages inhibit Th17 cell development. Therefore, one has construed that osteoclastogenesis is inhibited by the same Th1, Th2, and Treg lineage signature cytokines counteracting the stimulatory po‐ tential of the cytokine mixture secreted by the Th17 cells (Adamopoulos, Sabokbar et al. 2006; Adamopoulos and Bowman 2008). Contrastingly, when comparing clonal cells lines,

**4.2. T-helper 17 cells in Rheumatoid Arthritis (RA)**

486 Regenerative Medicine and Tissue Engineering

**Figure 5. A**. Effect of microRNA manipulations and cytokines on human osteochondral cells and osteoclasts injected into the tibial muscle of SCID mice. X-ray and tissue histology of the lower leg (*m. tibialis*) from SCID mice. Each tibial muscle was injected with hMSCs (2.5 million cells) and osteoclasts (0.5 million cells). Yellow, blue and red stars indicate that X-ray analyses and histology (visual parameters) were not significantly altered when cells were manipulated by cytokines (IL-1, IL-6, IL-17, and TNFα). However, molecular parameters were dramatically changed. X-ray analyses and histology were performed on excised legs 21 days subsequent to cell deposition. **B**. Differentiated chondrocytes (21 days) exposed to cytokines (initial 14 days) or pre-microRNAs (initial 14 days) and osteoclasts injected into the tibial muscle of SCID mice. Molecular markers of chondrocytes and osteoclasts (PBMCs differentiated for 10 days) were ana‐ lysed by Q-PCR or colorimetric methods (GAG = glucosaminoglycan/DNA). Nd = Not determined. All values are given relative to control conditions (chondrocytes alone or chondrocytes + osteoclasts). **C**. Differentiated osteoblasts (21 days) exposed to cytokines (initial 14 days) or antago-microRNAs (initial 14 days) and osteoclasts were injected into the tibial muscle of SCID mice. Molecular markers of osteoblasts and osteoclasts (PBMCs differentiated for 10 days) were analysed by Q-PCR or colorimetric methods (Ca2+-analyses in HCl-extracts). All values are given related to control conditions (osteoblasts alone or osteoblasts + osteoclasts).
