Nerve growth factor negatively regulates bone marrow granulopoiesis

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Nerve growth factor negatively regulates bone marrow granulopoiesis during small intestinal inflammation

The regulation of haematopoiesis during inflammation is the subject of intense study with respect to anti-tumour immunity and chronic disease. In particular, the generation of neutrophils is crucial for host survival, yet the restoration of steady state haematopoiesis is also essential for return to health. It is known that neutrophil homoeostasis is positively regulated by Toll-like receptor signalling and inflammatory cytokines (Wirths et al, 2014), but the negative regulation of ‘emergency’ granulopoiesis is still poorly understood (Manx & Boettcher, 2014). Bone marrow stromal cells have been shown to both synthesize nerve growth factor (NGF) and express a functional p75NTR/TrkA receptor, suggesting that NGF is involved in the regulation of haematopoiesis (Tomellini et al,2014). Current literature presents conflicting evidence concerning the effect of NGF on myeloid progenitor colonies and their differentiation into mature cells. We hypothesized that NGF, a known survival factor for granulocytes, could also play a role in emergency granulopoiesis. Here we use a parasite infection model, Trichinella spiralis, to study an on-demand yet naturally resolving haematopoietic response during acute small intestinal inflammation (Pennock & Grencis, 2004). It has been shown previously that NGF is expressed by gut epithelium (Torrents et al, 2002) during T. spiralis infection. We were interested in the effect that systemic NGF may have on myelopoiesis in vivo during acute gut inflammation and, in particular, any effects on the generation of granulocytes, as these are thought to contribute to parasite survival and the control of inflammation. During infection we found that there was no significant difference in either total monocytic (CD11b+Ly6GCD115+) ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 175, 161–175

or granulocytic (Ly6G+) cell types in the bone marrow, although a trend towards reduced monocyte numbers could be seen. However, gut homing neutrophils (CD11b+Ly6G+CD115-a4b7+) were significantly increased in the bone marrow (P < 0
01) at day 1 post-infection and in the blood at day 7 post-infection (P < 0
01, Fig 1A, B). This population was CD11blo/intLy6Gint, suggesting an immature phenotype similar to granulocytic myeloid-derived suppressor cells (Gr-MDSC) described previously (Van Ginderachter et al, 2010). Napthol AS-D chloroacetate staining of jejunum showed granulocytes were present the small intestine at day 7 post-infection (Fig 1C ii) correlating with the egress of gut homing cells from the bone marrow. Interestingly, infection induced a reduction in the ability of myeloid-committed progenitor cells to respond to ex vivo growth factors in every colony type, most significantly for granulocyte (CFU-G) and granulocyte/ macrophage colony-forming units (CFU-GM) (Fig 1D). When incubated with whole bone marrow from uninfected NIH mice, serum from infected animals reproduced the observed in vivo inhibition of myelopoiesis (Fig 1E), confirming the presence of systemic inhibitory factors and suggesting a dose response. These effects were inhibited by neutralizing anti-NGF antibody (Fig 1F) and could be replicated by coculture of na€ıve bone marrow for 7 days with exogenous recombinant NGF (1–500 ng/ml), causing a dose responsive inhibition of CFU-G (P < 0
001, data not shown). Of note, inhibition of serum NGF did not rescue CFU-M, suggesting that NGF acted primarily on the granulocyte lineage. To test the role of NGF in granulopoiesis in vivo during immunological challenge, T. spiralis infected mice were treated with a neutralizing anti-NGF antibody. Treatment induced 163

Correspondence

Fig 1. Granulocytes generated during Trichinella spiralis infection are regulated by nerve growth factor. (A & B) Myeloid bone marrow and blood cells were identified using flow cytometry. Granulocytes were gated as CD11b+CD115Ly6G+ to exclude monocytic cells (A). Gut homing CD11b+CD115Ly6G+a4b7+ cells peaked alternately in the bone marrow (black bars) and blood (grey bars) during infection (B). (C) Granulocytes could be seen by napthol AS-D chloroacetate staining of jejunal paraffin sections from infected mice at day 7 post-infection (ii) compared to uninfected mice (i). Inset (iii) represents optical zoom for clarity. Scale bar = 100 lm. (D) Bone marrow was removed and cultured ex vivo at the specified time points. Infection reduced the colony forming unit (CFU) proliferative potential of myeloid progenitors, most significantly for GFU-G and CFU-GM. (E) Serum from T. spiralis infected mice was incubated with na€ıve bone marrow and distinct CFUs were counted. Incubation of na€ıve bone marrow with serum from infected mice induced a steady loss in total CFU-G. (F) Anti-NGF neutralizing antibody ablated effects on CFU-G when incubated with infected serum. **P < 0
01 Data shown  SEM. (A–D) Data representative of three independent experiments (n = 3–6). (E & F) Data representative of two independent experiments (n = 5).

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ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 175, 161–175

Correspondence

Fig 2. Anti-NGF treatment increased overall granulocytic response in vivo during T. spiralis infection. (A) Granulocytes, as shown by napthol AS-D chloroacetate esterase staining, were significantly increased in the anti NGF group at day 12 post-infection (*P < 0
05). (B–E) Whole bone marrow was analysed by colony forming unit (CFU) and flow cytometry as previously described in the legend to Fig 1. (B) Anti-NGF treatment (dark bars) increased the production of total CFU-G by 4-fold in the bone marrow when compared to control mice (light bars) (ANOVA P < 0
001). (C–E) Representative flow cytometry plots at day 5 post-infection. Anti NGF treatment induced a distinct population of CD11b+ cells (C) and broad expression of a4b7+ (D) in the bone marrow when compared to phosphate-buffered saline controls. (E) CD11b+Ly6G+CD115a4b7+ cells were primarily CD11blo, suggesting immature Gr-MDSC. Data shown  SEM, representative of two independent experiments (n = 5).

significant weight loss (9
7%, P < 0
001) and small intestinal villus hyperplasia at day 12 post-infection (P < 0
001). These observations correlated with an increase in the overall granulocytic response: significantly more granulocytes were observed in the inflamed gut at day 12 post-infection (P < 0
05, Fig 2A); a 4-fold increase in CFU-G (P < 0
001, Fig 2B) in the bone marrow and a significant increase in gut homing granulocytes (CD11b+CD115Ly6G+a4b7+) was also seen in the bone marrow at day 5 post-infection (P < 0
05). Interestingly, the proportion of a4b7+ granulocytic myeloid cells did not ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 175, 161–175

change despite an overall increase in a4b7+ (Fig 2D), demonstrating an overall stimulation of granulopoiesis (reflected by an increase in the total pool of CD11b+ cells, Fig 2C), rather than a skew towards gut-specific cell types. Importantly, there was no change in the overall percentage of gut homing monocytic cells (CD11b+Ly6GCD115+ a4b7+) with inhibition of NGF, demonstrating a specific effect on granulocytic progenitors. Finally, 100% of the CD11b+Ly6G+CD115 cells that expressed a4b7 were CD11blo (Fig 2E) demonstrating that gut homing cells from the granulocytic pool maintained their 165

Correspondence immature Gr-MDSC phenotype after treatment (Van Ginderachter et al, 2010). It is known that functional subtypes of neutrophil exist, (Kruger et al, 2015) and this is particularly apparent in parasitic infection where they have been shown to be both antiparasitic (Saleem et al, 2012) and immunosuppressive (Van Ginderachter et al, 2010). Here we show clearly that during T. spiralis infection gut homing neutrophils are reminiscent of previously defined Gr-MDSC (Van Ginderachter et al, 2010) and that they arise in the bone marrow. Paradoxically, NGF has been shown to promote survival and function of inflammatory neutrophils, but also to be protective during acute gut inflammation (Reinshagen et al, 2000). Here we have shown that NGF is necessary to downregulate the proliferative response of bone marrow granulocytic progenitors both in vitro and in vivo. Our data suggest that the decline of committed granulocytic progenitor proliferation is a dosedependent response to the increase in systemic NGF and is independent of any effect on CFU-M. These data demonstrate that NGF is an important systemic regulatory factor during the resolution of gut inflammation, promoting survival of peripheral neutrophils whilst regulating their production in the bone marrow. However although NGF is sufficient for the regulation of granulocytes during acute gut inflammation, it is not necessary for gut homing. We suggest therefore that NGF may play an important role in granulopoiesis in other inflammatory conditions, such as allergy and psoriasis (Nockher & Renz, 2006).

References Kruger, P., Saffarzadeh, M., Weber, A.N., Rieber, N., Radsak, M., von Bernuth, H., Benarafa, C., Roos, D., Skokowa, J. & Hartl, D. (2015) Neutrophils: between host defence, immune modulation, and tissue injury. PLoS Pathogen, 11, e1004651. Manx, M.G. & Boettcher, S. (2014) Emergency granulopoiesis. Nature Reviews Immunology, 14, 302–314. Nockher, W.A. & Renz, H. (2006) Neurotrophins in allergic diseases: from neuronal growth factors to intercellular signaling molecules. Journal of Allergy and Clinical Immunology, 117, 583–589. Pennock, J.L. & Grencis, R.K. (2004) In vivo exit of c-kit+/CD49d(hi)beta7 + mucosal mast cell

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In conclusion, this work highlights the balance between local damage, immune responsiveness and haematopoietic regulation. Understanding the regulation of granulopoiesis and the generation of specific subpopulations will have a huge impact on understanding steady state haematopoiesis, acute inflammation and chronic inflammatory diseases. Finally, this work demonstrates the continuous cross talk between the nervous and immune systems and the pleiotropic role for each in the induction and regulation of inflammation. Swapna V. Vaddi1 Sayema Rahman2 Bakri M Assas3,4 Joanne L. Pennock4 Jaleel Miyan2 1 Evotek (UK) Ltd, Manchester, UK, 2Faculty of Life Sciences, University of Manchester, Manchester, UK, 3Faculty of Applied Medical Sciences,

King AbdulAziz University, Jeddah, Saudi Arabia and 4Institute of Inflammation & Repair, Faculty of Medicine and Human Sciences, University of Manchester, Manchester, UK E-mail: [email protected]

Keywords: haematopoiesis, granulocyte, immunology, progenitor cells First published online 18 December 2015 doi: 10.1111/bjh.13839

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