Transected axons typically fail to regenerate in the central nervous system (CNS), resulting in chronic neurological disability in individuals with traumatic brain or spinal cord injury, glaucoma and ischemic reperfusion injury of the eye. Although neuroinflammation is often depicted as detrimental, there is growing evidence that alternatively activated, reparative leukocyte subsets and their products can be deployed to improve neurological outcomes. In the current study we identify a unique granulocyte subset, with characteristics of an immature neutrophil, that had neuroprotective properties and drove CNS axon regeneration in vivo , in part via secretion of a cocktail of growth factors. This pro-regenerative neutrophil promoted repair in the optic nerve and spinal cord, demonstrating its relevance across CNS compartments and neuronal populations. Our findings could ultimately lead to the development of novel immunotherapies that reverse CNS damage and restore lost neurological function across a spectrum of diseases.

Interleukin-2 (IL-2) and anti-IL-2 antibody immune complex has recently been shown to expand the naturally occurring pool of CD4+Foxp3+ regulatory T cells (Foxp3+ Tregs). In this report, we showed that administration of IL-2/anti-IL-2 antibody immunocomplex to C57BL/6 mice, prior to corneal herpes simplex virus-1 (HSV-1) infection, significantly increased the pool of Foxp3+ Tregs when measured at early time-points post-infection. Increased numbers of Foxp3+ Tregs on day 2 and day 4 post-infection resulted in a marked reduction in the development of severe HSK. When compared to corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 post-infection. Reduced viral load was associated with two-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 post-infection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6 and 7 post-infection significantly increased Foxp3+ Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 post-infection. Our findings demonstrate that increasing Foxp3+ Tregs early but not late after infection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.

Substance P neuropeptide and its receptor neurokinin-1 (NK1R) are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation, increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells, and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional dendritic cells (cDCs) was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R−/− mice. After ocular HSV-1 infection, the number of cDCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R−/− than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R−/− mice. As a result, the number of IFN-γ secreting virus specific CD4 T cells in the DLNs of NK1R−/− mice was much higher than infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R−/− mice was associated with an early development of severe HSK. Collectively, our results show that the altered corneal biology of uninfected NK1R−/− mice along with an enhanced immunological response after ocular HSV-1 infection cause an early development of HSK in NK1R−/− mice.

Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Psbetaeudomonas (P.) aeruginosa infection of the cornea in BALB/c mice does not result in perforation and the mice have been classified as resistant. However, regulation of this response via inflammasome activation remained untested. Therefore, BALB/c mice were infected with P. aeruginosa ATCC strain 19660 and NLRP3 and NLRC4 protein tested by ELISA. Since NLRC4 vs NLRP3 protein levels were significantly higher in the corneas of BALB/c at 1 and 5 days postinfection we used silencing to knockdown NLRC4. Silencing NLRC4 vs scrambled siRNA treatment exacerbated disease in BALB/c mice, reduced myeloperoxidase levels and elevated bacterial plate counts at 5 days postinfection. It also increased pro IL-1beta, but reduced total protein for IL-1beta and IL-18 at 5 days postinfection. Flow cytometry to identify cells affected by silencing, showed reduced caspase-1 levels in a CD11blowLy6Glow population of cells, (but not PMN or macrophages) from the infected cornea of siNLRC4 treated mice that produced less mature IL-1beta. These data provide evidence that the NLRC4 inflammasome contributes to resistance through regulation of caspase-1, IL-1beta and IL-18 in a CD11blowLy6Glow population of cells.

Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing-remitting MS typically presents in the third or fourth decade, whereas the mean age of presentation of progressive MS (PMS) is 45 years old. Here, we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4 + Th17 cells, was more severe, and less likely to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils were more abundant, while B cells were relatively sparse, in CNS infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrated that radio-resistant, nonhematopoietic cells played a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of PMS, EAE in middle-aged adoptive transfer recipients was characterized by widespread microglial activation. Microglia from older mice expressed a distinctive transcriptomic profile suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.

The goal of this study was to determine the role of insulin-like growth factorbinding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). METHODS. In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro-and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3 −/−) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. RESULTS. Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3 −/− than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3 −/− than B6 mice. CONCLUSIONS. Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.