*2.4. Gene Expression Changes Associated with Chronic Antibiotic Treatment*

To determine antibiotic treatment-related molecular changes in knee joints we compared the transcriptome of 10-week-old mice that had been treated with AB in their drinking water to VEH controls. We found 620 significantly upregulated genes in uninjured AB compared to the uninjured VEH group; the majority of genes accounting for these transcriptional changes were associated with immune responses. Some of the groups we found were of genes associated with collagen (*C1qb* [44], and *Fcna* [45]), regulators of adaptive immunity (*Btla* [46], *Lax1* [47], *Tnfrsf13c* [48], *Lat* [49], and *Cd40* [50]), and genes associated with the major histocompatibility complex type II (*Tnfrsf14* [51], *Cd86* [52], and *Cd8b1* [53]). We found 737 significantly downregulated genes in the AB uninjured group compared to the VEH uninjured group. These genes included regulators of skeletal development (*Alpl* [37], *Col6a1* [54], *Col6a2* [55], *Sox9* [37], *Sox11* [56], and *Wnt9a* [57]), Hippo signaling (*Tead1*, *Tead4* [58], and *Yap1* [59]), muscle contraction (*Myh8*, *Myh2*, and *Myom2*), and inflammatory response (*Tlr5* [60], *Ccl8* [61], *Cxcl13* [37], *C5ar1* [62], *Cxcr1* [63], and *Foxo6* [64]). Inflammatory gene comparison between AB injured and uninjured groups compared to the corresponding VEH groups are shown in Figure 5A and Table 1.

**Figure 5.** Gene expression changes associated with antibiotic treatment. (**A**) Genes associated with inflammatory process modified by AB treatment and AB with injury compared to VEH uninjured and injured. (**B**) Genes associated with bone and cartilage formation modified by AB treatment along with injury compared to VEH uninjured and injured.


**Table 1.** Inflammatory gene overlap between the AB and VEH groups, showing the expression levels. Fold change (log2 scale) values are shown in the table (FDR < 0.05 for all genes). Changes that were not significant are denoted as ns.

We also found 185 genes significantly upregulated when comparing the AB injured to the VEH injured group. Among these we found negative regulators of cytokine production (*Arg1* and *Sars*) and small molecule catabolic processes (*Bad* and *Galk1*). We found 284 genes significantly downregulated when comparing the AB injured group to the VEH injured group. Among these we found regulators of the Wnt signaling pathway (*Sost* [35,65], *Fzd3*), Notch signaling pathway (Notch4), collagen-associated genes (*Chad*, *Col16a1*, and *Lep*), and transcription factors known to be involved in cellular differentiation processes (*Sox18*, *Foxd2*, and *Sox10*). Genes associated with bone and cartilage formation are shown in Figure 5B and Table 2.

**Table 2.** Bone and cartilage gene overlap between the AB and VEH groups, showing expression levels. Fold change (log2 scale) values are shown in the table (FDR < 0.05 for all genes). Changes that were not significant are denoted as ns.


There were 78 genes upregulated in both AB groups when compared to their corresponding VEH control groups, including *C1qb* [44] and *Cd3d* [66]. There were 151 genes downregulated in both AB groups compared to the VEH group. These included skeletal muscle contraction (*Myh1*, *Myh3* [67], *Myh14* [68], and *Myom3* [69]), and extracellular matrix proteins found in bone and cartilage (*Col1a1*, *Col2a1*, *Col5a1*, *Col6a3* [37], and *Col7a1* [70]). *Tbx5* and *Klhl40* were upregulated in the AB injured group and downregulated in the AB uninjured group when compared to their corresponding VEH groups. *Cd209a* and *Rspo1* were downregulated in the AB injured group and upregulated in the AB uninjured group when compared to their corresponding VEH groups. Comparing the AB uninjured and AB injured groups showed that bone formation genes like *Cthrc1* were upregulated in the AB injured group. *Mmp3* [37], *Lox*, *Loxl3*, *Mmp10*, and *Mmp19*, were downregulated in the AB uninjured group when compared to the AB injured group.

Comparison of Gene Expression Changes Between Chronic Antibiotic Treatment and LPS Treatment

In order to compare treatment-related molecular changes in the joint we compared the expression of AB and LPS to VEH of the same injury. We found 32 genes that were upregulated in both the AB and LPS groups compared to the uninjured VEH group. Among these we found genes related to the inflammatory and immune system (*Cx3cr1*, *Cd3d*, *Ccl4*, and *Ccl6*). We found *Rspo1* [71] to be upregulated in both the AB and LPS groups compared to the VEH groups, and downregulated in the AB injured group when compared to the injured VEH group. There were three genes (*Fgfbp1*, *Tnnc1*, and *Rab20*) found to be upregulated in the injured AB and LPS groups compared to the injured VEH. We found 18 genes downregulated in the AB and LPS uninjured groups when compared to the corresponding VEH groups. Genes related to the immune system were among these (*Bcl3*, *Prtn3*, *Tnfsf9*, and *Ifitm1*). We found seven genes to be upregulated in the uninjured LPS group while downregulated in the uninjured AB group when compared to the corresponding VEH groups. These genes are *Tlr5*, *C5ar1*, *Aqp4*, *Ryr3*, *Mdga1*, *Foxo6*, and *Kcng4*. We found two genes (*Cd209a* and *Lep*) downregulated in both injured the AB and LPS groups compared to the injured vehicle. We found marker *Cd209a*, which is present in macrophages and dendritic cells, to be upregulated in the uninjured AB and LPS groups and downregulated in the injured AB and LPS groups when compared to the corresponding VEH groups. Gene expression between the AB and LPS groups compared to the VEH groups is found in Table 3.

**Table 3.** Overlap of upregulated genes between the AB and LPS groups compared to the VEH showing the expression levels. Fold change (log2 scale) values are shown in the table (FDR < 0.05 for all genes). Changes that were not significant are denoted as ns.



**Table 3.** *Cont.*
