*2.3. LPS Treatment Compared to AB Treatment*

Lipopolysaccharides (LPSs) administered five days prior to the joint injury did not elicit any significant changes in the bone phenotype when compared to the AB-treated uninjured bones. However, as previously reported [10], LPS administration alone was sufficient to modulate the cartilage phenotype on both the contralateral and injured joints towards a more severe phenotype (Figure 3E,F). LPS injured joints showed an enhanced thinning of the femoral cartilage that was distinguishable from the cartilage of the VEH and AB injured groups (Figure 3b,d,f). The damage to the cartilage in the LPS injured joints corresponded with a significantly higher OARSI score than the VEH and AB injured joints (Figure 3I). There was an increase in cellular infiltration on the AB- and LPS-treated joints compared to the VEH (Figure 3bb,dd,ff). Combination of the AB treatment and LPS challenge (AB+LPS) significantly improved the LPS-mediated phenotype, reverting the contralaterals back to the OARSI scores recorded for the VEH injured and uninjured groups (Figure 3A,B,G,H). These results indicate that the effects of LPSs are blunted by the AB treatment, where statistical analysis does not distinguish between VEH and AB+LPS in either the uninjured or injured groups (Figure 3I).

**Figure 3.** Characterization of PTOA-associated structural changes in the injured VEH, AB, LPS, and AB+LPS groups. (**A**–**hh**) Histological evaluation of uninjured and injured joints at six weeks post injury using Safranin-O and Fast Green staining. Black scale bars indicate 200 μm. Numeral sign indicates the femoral condyle (**A**,**C**,**E**,**G**). High magnification images corresponding to yellow boxes (**B**,**D**,**F**,**H**) are provided (**b**, **bb**, **d**, **dd**, **f**, ff, **h**, **hh**). Arrows and asterisks indicate the thickness of the cartilage in the femoral condyle (**b**, **d**, **f**, **h**). Asterisks indicate cellularity in the synovium (**bb**, **dd**, ff, **hh**). (**I**) OARSI scores (\* *p*-value < 0.05).

Macrophages Associated with Healing Are Increased in Antibiotic-Treated Joints

Tissue resident macrophages are essential in providing innate immune defenses and regulating tissue and organ homeostasis [39]. Macrophages and other inflammatory cells are recruited to injury sites where they also play key roles in tissue remodeling and repair [40,41]. In the joint there is a population of inactive macrophages residing in the synovium [42]. These cells are activated under certain conditions such as injury or inflammation. Macrophages can be found using the marker F4/80, which is a marker for cells of mononuclear phagocyte lineage in mice [43]. Macrophages can be both pro-inflammatory (M1) and anti-inflammatory (M2), and the co-action of these subtypes can repair damaged tissue through specific cytokine secretion. Identification of both macrophage populations was

done using F4/80 and iNOS as an M1 marker while using CD206 as a M2 marker. In the joint, however, a change in the M1/M2 ratio may be critical in PTOA progression and development. Histological examination of the injured joints indicated a hyperplastic synovium that appeared to have significant cellular infiltration on LPS- and AB-treated injured joints (Figure 3dd,ff; asterisk), and this morphology was similar to that of LPS-treated injured joints previously described [10]. To determine whether AB treatment prior to ACL rupture altered the composition of M1 and M2 cells in the injured joint, we used M1/M2 specific antibody markers to distinguish these subtypes by immunohistochemistry (Figure 4). On the meniscus, we observed a slightly higher staining of anti iNOS antibody indicative of some M1 macrophage infiltration in the AB injured joints compared to the VEH injured joints (Figure 4A,B). The M1 macrophage infiltration presence in AB injured joints was less than that of LPS injured joints (Figure 4B,C). In contrast, we observed a higher level of anti CD206 antibody staining in AB injured joints when compared to VEH and LPS injured, indicative of higher levels of M2 macrophages present in the injured joints of AB-treated animals (Figure 4D–F). These results suggest that while there is an increase in macrophages for both LPS and AB treatments, the LPS-treated joints have an increase in pro-inflammatory macrophages while AB-treated joints have an increase in anti-inflammatory macrophages that may be helping mitigate the PTOA phenotype.

**Figure 4.** Macrophage infiltration analysis of the VEH-, AB-, and LPS-treated injured joints. (**A**–**C**) Fluorescent immunohistochemistry (IHC) of macrophage type 1 markers F4/80 and iNOS. (**D**–**F**) Fluorescent IHC of macrophage type 2 markers F4/80 and CD205. Dashed line in white represents the surface of the anterior tibial condyle for all images. (**a**–**f**) DAPI; (**aa**–ff) F4/80; (**aaa**–**ccc**) iNOS; (**ddd**–ff**f**) CD206. White scale bar represents 100 μm.
