Heme-Induced Macrophage Phenotype Switching and Impaired Endogenous Opioid Homeostasis Correlate with Chronic Widespread Pain in HIV
and Saurabh Aggarwal 1,*Round 1
Reviewer 1 Report
The MS by Chatterjee et al. investigates the potential role of cell-free heme in the polarization of macrophages and the effects on endogenous opioid synthesis and release. The authors propose that heme-dependent TLR4 activation polarizes macrophages to M1 phenotype, resulting in lower amounts of released β-endorphin than M2 phenotype macrophages, which could potentially contribute to chronic widespread pain in people living with HIV. The paper is well-structured and clearly presents the research question and hypothesis, followed by a detailed results description. The authors use a combination of in vitro and in vivo approaches. The results of the study, however, seem to be not highly novel: heme-induced TLR4 activation, macrophage polarization and decreased β-endorphin production were shown before (PMID: 17502383, PMID: 32828015, PMID: 26675351). The authors should clearly describe which findings are novel in the current paper. Overall, I find the results of the study convincing, while still leaving some areas to improve.
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Please, re-write the abstract in a way that it would be clear what is the study background and what was done novel in the current paper.
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Why were CD4+ T cell levels not reported for the HIV-negative population?
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Please, provide the details of cART regimen in HIV-positive groups (type of therapy, duration and address potential differences between pain vs. no pain groups).
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Gating strategy on FSC-SSC plot doesn't seem correct on Fig. S1A and is absent in Fig. S2A.
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The authors can discuss the potential causes of the high heme rates in people living with HIV with chronic widespread pain.
Minor remarks:
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Table 1. Please, use also a standard annotation for p-values to describe the level of significance for each comparison (* p < 0,05, ** p < 0.01, etc.)
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The authors should re-read the paper for some typos. The examples include:
L. 252-254 The sentence is unclear.
L. 263 - no individual values are present
Author Response
The MS by Chatterjee et al. investigates the potential role of cell-free heme in the polarization of macrophages and the effects on endogenous opioid synthesis and release. The authors propose that heme-dependent TLR4 activation polarizes macrophages to M1 phenotype, resulting in lower amounts of released β-endorphin than M2 phenotype macrophages, which could potentially contribute to chronic widespread pain in people living with HIV. The paper is well-structured and clearly presents the research question and hypothesis, followed by a detailed results description. The authors use a combination of in vitro and in vivo approaches. The results of the study, however, seem to be not highly novel: heme-induced TLR4 activation, macrophage polarization and decreased β-endorphin production were shown before (PMID: 17502383, PMID: 32828015, PMID: 26675351). The authors should clearly describe which findings are novel in the current paper. Overall, I find the results of the study convincing, while still leaving some areas to improve.
- Please, re-write the abstract in a way that it would be clear what is the study background and what was done novel in the current paper.
We have revised the abstract as suggested by the reviewer.
Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis, elevated plasma levels of cell-free heme, which correlated with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs β-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens β-endorphin production are inconclusive. Current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to M1 phenotype mediates this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to M1 phenotype with low β-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased M1/M2 cell ratio and a reduced β-endorphin levels. However, treating these animals with the heme scavenging protein, hemopexin (Hx) or TAK-242, reduced M1/M2 ratio and increased β-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlates with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV.
- Why were CD4+ T cell levels not reported for the HIV-negative population.
CD4 counts are not routinely measured in HIV negative participants.
- Please, provide the details of cART regimen in HIV-positive groups (type of therapy, duration and address potential differences between pain vs. no pain groups).
Detailed information about cART has been added in the methodology section (lines 93-99).
Majority of HIV-positive participants at the UAB HIV clinic were on intregrase inhibitor (bictegravir, dolutegravir) based regimens paired with 2 nucleoside reverse transcriptase inhibitors (NRTI) (tenofovir, alfenomide). A smaller portion were on non-nucleoside reverse transcriptase inhibitor (NNRTI) (doravirine, rilpivirine) based regimens paired with 2-NRTI’s. Patients were on these therapies for at least 5 years and there were no significant differences with regard to anti-retroviral therapies between people with HIV with pain and without chronic pain.
- Gating strategy on FSC-SSC plot doesn't seem correct on Fig. S1A and is absent in Fig. S2A.
We have modified Fig. S1A and added the gate in Fig.S2B as requested.
- The authors can discuss the potential causes of the high heme rates in people living with HIV with chronic widespread pain.
We have now added discussion about most relevant causes of hemolysis in HIV in discussion (lines 484-493).
PWH have higher prevalence of intravascular hemolysis than HIV negative individuals. Some of the reported mechanism of hemolysis in HIV include low red blood cell (RBC) glutathione, an anti-oxidant required to maintain the normal structure of RBCs[34-36]. Anti- retroviral therapy or other drugs for HIV-associated infections such as the use of amphotericin B and co-trimoxazole for cryptococcal meningitis and the use of interferon and ribavirin for hepatitis C may also increase the risk of hemolysis[37-39]. HIV also confers 15–40-fold higher risk of acquired thrombotic microangiopathy compared with the HIV negative population, which is associated with increased intravascular hemolysis[40-42]. PWH also have higher prevalence of glucose 6 phosphate dehydrogenase (G6PD) deficiency, which is an important cause of hemolysis[43].
Minor remarks:
- Table 1. Please, use also a standard annotation for p-values to describe the level of significance for each comparison (* p < 0,05, ** p < 0.01, etc.)
We incorporated annotation for p-values as suggested.
- The authors should re-read the paper for some typos. The examples include:
- 252-254 The sentence is unclear.
We made suggested corrections in the revised manuscript.
- 263 - no individual values are present.
We have made the correction as suggested.
Reviewer 2 Report
The manuscript entitled “Heme-induced macrophage phenotype switching is associated 2 with low endogenous opioids and chronic widespread pain in 3 HIV" appears to be interesting, but there are many flaws and concerns on it. Study can be greatly improved if following suggestions were incorporated.
1. The title of the paper is not accurately expressed, and I think it needs to be rewritten.
2. Some references missing.
3. There are many grammatical corrections, the authors are expected to incorporate those corrections as well.
4. In order to make the paper more interesting to read, I suggested that the authors could add one graphical abstract to the manuscript.
5. I suggest including clear limitations of the study in the discussion.
There are many grammatical corrections, the authors are expected to incorporate those corrections as well.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
