Skeletal muscle repair and regeneration after injury requires coordinated interactions between the innate immune system and the injured muscle. Myeloid cells predominate in these interactions. This study examined the role of KLF2, a zinc-finger transcription factor that regulates immune cell activation, in specifying myeloid cell functions during muscle regeneration. Loss of KLF2 in myeloid lineage cells (myeKlf2-/- mice) dramatically enhanced the initial inflammatory response to acute muscle injury (cardiotoxin). Injured muscles showed dramatically elevated expression of inflammatory mediators and greater numbers of infiltrating, pro-inflammatory monocytes that matured earlier into activated macrophages. Notably, the inflammatory phase resolved earlier and regeneration progressed to myogenesis, marked by elevated expression of factors that promote the formation of new fibers from satellite cells. Regeneration was completed earlier, with phenotypically normal adult fibers integrated into the muscle syncytium. These findings identify myeloid KLF2 as a key regulator of myeloid cell functions in adult skeletal muscle regeneration.

During chronic intestinal inflammation in rabbit intestinal villus cells brush border membrane (BBM) Na-glucose co-transport (SGLT1), but not Na/H exchange (NHE3) is inhibited. The mechanism of inhibition is secondary to a decrease in the number of BBM co-transporters. In the chronic enteritis mucosa, inducible nitric oxide (iNO) and superoxide production are known to be increased and together they produce abundant peroxynitrite (OONO), a potent oxidant. However, whether OONO mediates the SGLT1 and NHE3 changes in intestinal epithelial cells during chronic intestinal inflammation is unknown. Thus, we determined the effect of OONO on SGLT1 and NHE3 in small intestinal epithelial cell (IEC-18) monolayers grown on trans well plates. In cells treated with 100 μM SIN-1 (OONO donor) for 24 h, SGLT1 was inhibited while NHE3 activity was unaltered. SIN-1 treated cells produced 40 times more OONO fluorescence compared to control cells. Uric acid (1mM) a natural scavenger of OONO prevented the OONO mediated SGLT1 inhibition. Na⁺/K⁺-ATPase which maintains the favorable trans-cellular Na gradient for Na-dependent absorptive processes was decreased by OONO. Kinetics studies demonstrated that the mechanism of inhibition of SGLT1 by OONO was secondary to reduction in the number of co-transporters (V_max) without an alteration in the affinity. Western blot analysis showed a significant decrease in SGLT1 protein expression. Further, p38 mitogen-activated protein (MAP) kinase pathway appeared to mediate the OONO inhibition of SGLT1. Finally, at the level of the co-transporter, 3-Nitrotyrosine formation appears to be the mechanism of inhibition of SGLT1. In conclusion, peroxynitrite inhibited BBM SGLT1, but not NHE3 in intestinal epithelial cells. These changes and the mechanism of SGLT1 inhibition by OONO in IEC-18 cells is identical to that seen in villus cells during chronic enteritis. Thus, these data indicate that peroxynitrite, known to be elevated in the mucosa, may mediate the inhibition of villus cell BBM SGLT1 in vivo in the chronically inflamed intestine.

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

In the mammalian small intestine, sodium is primarily absorbed by Na⁺/H⁺ exchange (NHE3) and Na‐glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC‐18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC‐18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na‐extruding capacity of the cell since Na⁺/K⁺‐ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between the injured muscle and the innate immune system. Myeloid lineage cells predominate in this process. This study examined the role of Krüppel like factor 2 (KLF2), a zinc-finger transcription factor that regulates myeloid cell activation state, in muscle regeneration. Gastrocnemius muscles of wild-type and myeKlf2-/- mice, which lack KLF2 in all myeloid cells, were subjected to cardiotoxin injury and followed for 21 days. Injured muscles of myeKlf2-/- contained more infiltrating, inflammatory Ly6C+ monocytes, with elevated expression of inflammatory mediators. Infiltrating monocytes matured earlier into pro-inflammatory macrophages with phenotype Ly6C+, CD11b+, F4/80+. Inflammation resolved earlier and progressed to myogenesis, marked by an earlier decline of Ly6C+ macrophages and their replacement with anti-inflammatory Ly6C- populations, in association with elevated expression of factors that resolve inflammation and promote myogenesis. Overall, regeneration was completed earlier. These findings identify myeloid KLF2 as a central regulator of the innate immune response to acute skeletal muscle injury. Manipulating myeloid KLF2 levels may be a useful strategy for accelerating regeneration.