In the present work from Rai et al., titled “Counteractive effects of IL-33 and IL-37 on Inflammation in Osteoarthritis”, the authors investigate the interactive role of Interleukins 33 and 37 which have been previously involved in pro- and anti-inflamatory processes, respectively. The research is mainly based on immunofluorescence and RT-PCR techniques and includes a flow cytometry approach to study macrophage polarization.

I have several important major concerns about the manuscript on its present form:

1) First of all, the manuscript needs extensive English language edition. Some gramatically incorrect and intricated sentences must be corrected. Some examples are:

-Lines 67-70: “The involvement of macrophages in OA is suggested by the detection of activated macrophages mediating the progression of structural degeneration (CD163 and CD14) and pain (CD14) and soluble macrophage biomarkers as an indicator of inflammatory phenotypes in the OA knee joint and [26, 27].” So many “and” including the final one should be avoided.

Lines 73-77: “The role of the synovial macrophage in osteophyte formation and OA-related pathology [24], macrophage inflammatory protein 3α in rheumatoid arthritis (RA) and OA, and macrophage-derived pro-inflammatory cytokines in OA associate inflammatory macrophage (M1) with arthritis (RA and OA) [25]. “ This sentence is too long , the subject should be shortened.

Line 108: Check superindex.

Line 221: “H&E” or "HE" instead of “H and E”.

Lines 230-232: “A higher expression of IL-33 in the knee joint compared to the hip joint cartilage higher expression of IL-37 in the hip joint compared to the knee joint cartilage (Fig. 2) was revealed with immunofluorescence” So many “higher expression” and “compared” make the sentence confuse.

Line 268 and later: Please keep the same format for all the results subheadlines

Lines 295-297: “cDNA synthesized from NHAC cells treated with IL-37 (50 ng/ml) followed by IL-33 (25ng/ml), rHMGB-1 (500ng/ml), and LPS (100ng/ml) for 24 hours was subjected to RT-PCR” is a tricky/complicated way to introduce the result.

2) In the immunofluorescence results presented in Sup Fig 1 and 2:

• I assume that all the tissues collected from joint replacement are OA tissues. However, even in the same OA joint, different zones of OA progression can be differentiated, some times even macroscopically. Did the authors had this into account when collecting the samples? Have the authors tested the collagen II and aggrecan signals in different areas of the tissue?

-Do the authors mean Chitinase 3 or Chitinase 3 like protein 1?

-If they mean Chitinase 3 like protein 1, then why the fluorescence intensity in the OA tissues (Fig S1 D) is similar to the intensity in normal chondrocytes (Fig S2 M). Why is not as extense and diffuse than the signal in OA chondrocytes (Fig S2 P)?

-Lines 95-96: “Cartilage tissues were collected anonymously from patients under-95 going total knee or hip replacement due to severe osteoarthritis”. I hope not. Do you mean that samples and data were anonymized in the database?

3) What is the point of showing the Collagen II immunofluorescence signal in the OA tissues without showing the signal on a Normal tissue or at least a negative control?

In the chondrocytes, why collagen II expression is not investigated by RT-PCR to complement the immunofluorescence data? I am curious about the protein/mRNA levels correlation.

4) Lines 317-320: “Immunopositivity for CD14 (macrophage), CD86 (M1 macrophage), and CD206 (M2a macrophage) (Fig. 6) while minimal immunopositivity (data not shown) for CD163 (M2b macrophage) and IL-10 (M2c macrophage) was noted in post-surgical OA knee and hip joint cartilages using dual immunofluorescence”. That minimal immunopositivity data should be shown in the manuscript.

5) The information provided for the macrophage polarization determination is scarce. In the methodological section (2.10) there is no information on the flow cytometry instrument used for the analysis. In the results section some important information is missing, for instance:

Is the blue signal the negative signal from the isotype?

How is the negativity/positivity threshold selected?

Figure 7 quality is not acceptable for publication. Horizontal axes and units are confusing.

Why the positivity threshold used for instance for CD86 change along the horizontal axis depending on the stimulus (see first colum of panels)? This is specially confusing considering that the axis scale is the same in all the panels.