Reply to “Herpes Simplex Virus 1, Macrophages, and the Cornea”

REPLY

In a letter to the editor regarding our report in the Journal of Virology (JVI) (1), several issues were raised by Daniel Carr, and our responses to his points are as follows.

(i) Dr. Carr, in support of his comments, cites papers by Tumpey et al. (2) and Thomas et al. (3). However, in those studies, the authors examined the effect of neutrophil depletion on ocular herpes simplex virus 1 (HSV-1) infection and showed that depletion of neutrophils did not alter CD4⁻, CD8⁻, B-cell, NK cell, or F4/80 macrophage populations. No direct comparison of macrophages versus neutrophils populations in untreated and infected mice was done. In various human eye diseases, such as dry eye and ocular graft-versus-host disease, neutrophils were detected on the surface of the cornea (tears) but not inside corneal tissues (4, 5). Similarly, in both BALB/c and C57BL/6 mice, GR-1⁺ cells were not significant infiltrates and were not correlated with inflammation after corneal transplantation (6).

Similar to our results (1), Dr. Carr, in his own paper that was cited in the letter to JVI (7), reported more macrophages in the naive infected mouse cornea (approximately 3,000 cells) than neutrophils (approximately 1,250 cells) on day 5 postinfection (p.i.). Also, in another paper, Dr. Carr's group reported similar numbers of macrophages and neutrophils in the corneas of infected mice on day 30 p.i. (8). Dr. Carr also showed that neutrophils become dominant infiltrates in the corneas of HSV-1-infected mice after day 10 p.i. (9). In their discussion, they indicated that macrophages may play an important role in the early stages of infection. However, in our abstract and introduction, we were talking about primary ocular infection, which generally refers to days 1 to 7 p.i. and when infectious virus can be detected in the eye by plaque assays of an eye swab sample.

Many independent studies have clearly demonstrated that eye disease, infiltrates in the eye, latency, and reactivation all are affected by the strain of mouse, strain of virus, dose of infection, and infection with or without corneal scarification. All of these factors are possible reasons that there is even variability in Dr. Carr's own published studies. Dr. Carr's group used corneal scarification in their reported studies with very-low-dose infection (7, 9), and previously he has shown that corneal scarification is required for detection of HSV-1 in the retinas of infected mice (10). Thus, the presence of the virus in the retina, trauma associated with corneal scarification, and the variability of corneal scarification from experiment to experiment could very well affect the type of infiltrates in the corneas of infected mice. Consequently, results obtained after corneal scarification, which is a very traumatic procedure and needs complete anesthesia to be performed, does not reflect natural infection compared to infection without scarification, as we have done.

(ii) The other issue that was raised is in respect to resident macrophages. Corneal macrophages are classified as CCR2⁻ and CCR2⁺, and CCR2⁻ macrophages can be considered resident macrophages and are not affected by monocytes, while CCR2⁺ macrophages occupied the cornea under both normal and disease conditions (11). Migratory infiltrates following infection or trauma enter the affected site, as was reported previously (12). Dr. Carr, in his letter, also indicated that the cornea is a “pristine structure,” and previously we have shown detection of infiltrates after infection and not before infection by using immunohistochemistry and not fluorescence-activated cell sorter analyses in BALB/c mice (13–17).

(iii) Dr. Carr also discusses his published study using an ICP0-null virus similar to a virus that was originally developed in the laboratory of the late Priscila Shaffer (18). In the letter to JVI regarding our paper, Dr. Carr advertised the superiority of the vaccine used in his flawed study that he is marketing as a human vaccine through a company of which the author is a board member and received stock options. He compared his highly attenuated virus in an HSV-1 vaccine study with a gD2 subunit vaccine as a control rather than using gD1 as a control or another attenuated HSV-1 strain, which defies scientific logic and raises some serious questions about their findings and claims. Why compare HSV-1 vaccine against gD2 vaccine when gD1 would have been a better choice? However, we did not do a vaccination in our study, except insofar as to suggest that injection of mice with CSF-1 DNA is advantageous over gamma interferon (IFN-γ) DNA injection. Therefore, what is the relevance of his vaccination strategy to our injection strategy, except advertising for his commercial endeavor? In addition, we suggested in our discussion that if CSF-1 is included with a cocktail of HSV-1 glycoproteins, we may obtain better protection. In our paper, we clearly showed a shift in the phenotypes of macrophages after injection with IFN-γ DNA (toward M1) and CSF-1 DNA (toward M2), as well as higher levels of IFN-γ expression in the IFN-γ DNA-injected group. In addition, our results obtained with splenic and peritoneal macrophages from injected mice also clearly show a shift in systemic responses due to injection with CSF-1 DNA versus IFN-γ but not controls. Our study was specifically designed to look only at possible differences in M1 versus M2 macrophages both in vitro and in vivo. A more careful reading of our report and a better understanding of the subject would have eliminated these misconceptions.

(iv) Dr. Carr discusses a lack of statistical analysis for Tables 1 and 2. In those two tables, we looked at 34 cytokines and chemokines from six different treatments per cell type by using two different cell types. We purposely mentioned only those differences that we felt may be relevant to our model as a reference without including any statistical analysis so as not to clutter up the tables and to let readers draw their own conclusions. In addition, our statistical analyses are relevant to our experiments and are well established. Regarding analysis of variance (ANOVA), we believe that the two-tailed t test is an appropriate method, as we only compared two groups at a time. Also, we got the same results whether we used the Fisher exact test or ANOVA. Overall, our data were highly significant and we would have gotten the same results no matter what type of statistical analysis we used, and as one of my professors said many decades ago, “do not let statistics be your master.”

In conclusion, even Dr. Carr's own cited paper supports our studies (7). If Dr. Carr had paid more attention to how we performed the virus work, taken into account the complications associated with using corneal scarification to initiate infection of the eye, and realized that a very large dose of a virulent virus was used for this study, this unfortunate misinterpretation could have been avoided. To the best of our knowledge, our study was unique and novel and for the first time we correlated the in vitro function of M1 and M2 macrophages with their presence and function in vivo.